G-type peptides to ameliorate atherosclerosis

ABSTRACT

This invention provides novel peptides that ameliorate one or more symptoms of atherosclerosis and/or other pathologies characterized by an inflammatory response. In certain embodiment, the peptides resemble a G* amphipathic helix of apolipoprotein J. The peptides are highly stable and readily administered via an oral route.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This work was supported, in part, by Grant No: HL30568 from the NationalHeart Blood Lung Institute of the National Institutes of Health. TheGovernment of the United States of America may have certain rights inthis invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[Not Applicable]

FIELD OF THE INVENTION

This invention relates to the field of atherosclerosis. In particular,this invention pertains to the identification of a class of peptidesthat are orally administrable and that ameliorate one or more symptomsof atherosclerosis or other pathologies characterized by an inflammatoryresponse.

BACKGROUND OF THE INVENTION

The introduction of statins (e.g. Mevacor@, Lipitor@) has reducedmortality from heart attack and stroke by about one-third. However,heart attack and stroke remain the major cause of death and disability,particularly in the United States and in Western European countries.Heart attack and stroke are the result of a chronic inflammatorycondition, which is called atherosclerosis.

Several causative factors are implicated in the development ofcardiovascular disease including hereditary predisposition to thedisease, gender, lifestyle factors such as smoking and diet, age,hypertension, and hyperlipidemia, including hypercholesterolemia.Several of these factors, particularly hyperlipidemia andhypercholesteremia (high blood cholesterol concentrations) provide asignificant risk factor associated with atherosclerosis.

Cholesterol is present in the blood as free and esterified cholesterolwithin lipoprotein particles, commonly known as chylomicrons, very lowdensity lipoproteins (VLDLs), low density lipoproteins (LDLs), and highdensity lipoproteins (HDLs). Concentration of total cholesterol in theblood is influenced by (1) absorption of cholesterol from the digestivetract, (2) synthesis of cholesterol from dietary constituents such ascarbohydrates, proteins, fats and ethanol, and (3) removal ofcholesterol from blood by tissues, especially the liver, and subsequentconversion of the cholesterol to bile acids, steroid hormones, andbiliary cholesterol.

Maintenance of blood cholesterol concentrations is influenced by bothgenetic and environmental factors. Genetic factors include concentrationof rate-limiting enzymes in cholesterol biosynthesis, concentration ofreceptors for low density lipoproteins in the liver, concentration ofrate-limiting enzymes for conversion of cholesterols bile acids, ratesof synthesis and secretion of lipoproteins and gender of person.Environmental factors influencing the hemostasis of blood cholesterolconcentration in humans include dietary composition, incidence ofsmoking, physical activity, and use of a variety of pharmaceuticalagents. Dietary variables include amount and type of fat (saturated andpolyunsaturated fatty acids), amount of cholesterol, amount and type offiber, and perhaps amounts of vitamins such as vitamin C and D andminerals such as calcium.

Low density lipoprotein (LDL) oxidation has been strongly implicated inthe pathogenesis of atherosclerosis. High density lipoprotein (HDL) hasbeen found to be capable of protecting against LDL oxidation, but insome instances has been found to accelerate LDL oxidation. Importantinitiating factors in atherosclerosis include the production ofLDL-derived oxidized phospholipids.

Normal HDL has the capacity to prevent the formation of these oxidizedphospholipids and also to inactivate these oxidized phospholipids oncethey have formed. However, under some circumstances HDL can be convertedfrom an anti-inflammatory molecule to a pro-inflammatory molecule thatactually promotes the formation of these oxidized phospholipids.

HDL and LDL have been suggested to be part of the innate immune system(Navab et al. (2001) Arterioscler Thromb Vasc Biol. 21: 481-488). Thegeneration of anti-inflammatory HDL has been achieved with class Aamphipathic helical peptides that mimic the major protein of HDL,apolipoprotein A-I (apo A-I) (see, e.g., WO 02/15923).

SUMMARY OF THE INVENTION

This invention provides novel compositions and methods to amelioratesymptoms of atherosclerosis and other inflammatory conditions such asrheumatoid arthritis, lupus erythematous, polyarteritis nodosa,osteoporosis, Altzheimer's disease and viral illnesses such as influenzaA.

In certain embodiments this invention provides “isolated” polypeptidesthat ameliorate a symptom of atherosclerosis or other pathologiesassociated with an inflammatory response and/or compositions comprisingsuch polypeptides. The polypeptides typically comprise an amphipathichelical polypeptide having charged residues on the polar face of thepolypeptide and possessing a wide non-polar face. The polypeptide istypically at least about 10 amino acids in length and/or about 40 orfewer polypeptides in length. Preferred polypeptides typically comprisea G* amphipathic helix. In certain embodiments, the polypeptides showgreater than about 50%, preferably greater than about 75%, and morepreferably greater than about 85% sequence identity with apo J (e.g.over a domain the same length as the polypeptide in question). Preferredpolypeptides of this invention protect a phospholipid (e.g.,1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC),1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (SAPC)),1-stearoyl-2-arachidonyl-sn-glycero-3-phosphorylethanolamine (SAPE))against oxidation by an oxidizing agent (e.g., 13(S)-HPODE, 15(S)-HPETE,HPODE, HPETE, HODE, and HETE). Particularly preferred polypeptidescomprise or consist of one or more of the following amino acidsequences: LLEQLNEQFNWVSRLANLTEGE, (SEQ ID NO:1), LLEQLNEQFNWVSRLANL,(SEQ ID NO:2), NELQEMSNQGSKYVNKEIQNAVNGV, (SEQ ID NO:3), IQNAVNGVKQIKTLIEKTNEE, (SEQ ID NO:4), RKTLLSNLEEAKKKKEDALNETRESETKLKEL, (SEQ IDNO:5), PGVCNETMMALWEECK, (SEQ ID NO:6), PCLKQTCMKFYARVCR, (SEQ ID NO:7),ECKPCLKQTCMKFYARVCR, (SEQ ID NO:8), LVGRQLEEFL, (SEQ ID NO:9),MNGDRIDSLLEN, (SEQ ID NO:10), QQTHMLDVMQD, (SEQ ID NO:11),FSRASSIIDELFQD, (SEQ ID NO:12), PFLEMIHEAQQAMDI, (SEQ ID NO:13),PTEFIREGDDD, (SEQ ID NO:14), RMKDQCDKCREILSV, (SEQ ID NO:15),PSQAKLRRELDESLQVAERLTRKYNELLKSYQ, (SEQ ID NO:16), LLEQLNEQFNWVSRLANLTQGE, (SEQ ID NO:17), DQYYLRVTTVA, (SEQ ID NO:18),PSGVTEVVVKLFDS, (SEQ ID NO:19), PKFMETVAEKALQEYRKKHRE, (SEQ ID NO:20),WDRVKDLATVYVDVLKDSGRDYVSQF (SEQ ID NO:21), VATVMWDYFSQ LSNNAKEAVEHLQK(SEQ ID NO:22), RWELALGRFWDYLRWVQTLSEQVQEEL (SEQ ID NO:23),LSSQVTQELRALMDETMKELKELKAYKSELEEQLT (SEQ ID NO:24),ARLSKELQAAQARLGADMEDVCGRLV (SEQ ID NO:25), VRLASHLRKLRKRLLRD ADDLQKRLA(SEQ ID NO:26), PLVEDMQRQWAGLVEKVQA (SEQ ID NO:27), MSTYTGIFTDQVLSVLK(SEQ ID NO:28), and LLSFMQGYMKHATKTAKDALSS (SEQ ID NO:29). In certainembodiments, the polypeptide is a concatamer of two or more of theseamino acid sequences and/or a concatamer of one or more of these aminoacid sequences and an apo A-I sequence or a mimetic thereof (see, e.g.,PCT publication WO 02/15923 for apo A-I related polypeptides/mimetics).The polypeptides of this invention can comprise a protecting group (e.g.a protecting group on the amino and/or carboxyl terminus). Preferredprotecting groups include, but are not limited to acetyl, amide, 3 to 20carbon alkyl groups, Fmoc, t-boc, 9-fluoreneacetyl group,1-fluorenecarboxylic group, 9-florenecarboxylic group,9-fluorenone-1-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan),Trityl (Trt), 4-methyltrityl (Mtt), 4-methoxytrityl (Mmt),4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr), Mesitylene-2-sulphonyl(Mts), 4,4=-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethylchroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4-methoxybenzyl(MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene) ethyl (Dde),2,6-dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), a benzoyl group, acarbobenzoxy group, a propyl group, a butyl group, a pentyl group, ahexyl group, and Trifluoroacetyl (TFA). In certain embodiments, thepolypeptide comprises a protecting group coupled to the amino terminaland the amino terminal protecting group is a protecting group such as abenzoyl group, an acetyl, a propeonyl, a carbobenzoxy, a propyl, abutyl, a pentyl, a hexyl, or a 3 to 20 carbon alkyl. In certainembodiments, the polypeptide comprises a protecting group coupled to thecarboxyl terminal and the carboxyl terminal protecting group is anamide.

In particularly preferred embodiments, the polypeptide(s) of thisinvention comprise one or more dextro “D” amino acids. In certainembodiments, the polypeptide(s) of this invention comprise at least two,preferably at least 4, and most preferably all “D” amino acids.

In certain embodiments the polypeptide(s) described herein arecovalently coupled to a phospholipid (e.g. lysophosphatidyl choline). Inparticularly preferred embodiments, the polypeptide(s) are coupled tothe sn-1 or sn-2 position of a phospholipid (e.g. propionoyl, butanoyl,pentanoyl, caproyl, heptanoyl, capryloyl, nonanoyl, capryl, undcanoyl,lauroyl, tridecanoyl, myristoyl, pentadecanoyl, palmitoyl,heptadecanoyl, stearoyl, nonadecanoyl, arachidoyl, heniecosanoyl,behenoyl, trucisanoyl, lignoceroyl, myristoleoyl (9-cis), myristelaidoyl(9-trans), palmitoleoyl (9-cis), palmitelaidoyl (9-trans, and the like).

The polypeptide(s) of this invention can be formulated with apharmacologically acceptable excipient (e.g. a unit dosage formulationfor oral administration, rectal administration, nasal administration,injection, and the like).

In another embodiment, this invention provides a composition suitablefor oral administration that ameliorates a symptom of atherosclerosis orother pathologies characterized by an inflammatory response. Thecomposition comprises a polypeptide comprising an amphipathic helix(e.g. a G* helix) as described herein where the polypeptide comprisesone or more “D” amino acids as described herein and the polypeptide isblocked at the amino terminus and the carboxyl terminus as describedherein.

In certain embodiments, this invention provides pharmaceuticalformulations (compositions). The pharmaceuticals comprise a polypeptideas described herein in a pharmaceutically acceptable excipient. Theformulation is often a unit dosage formulation (e.g. for oral, rectal,nasal, or injectible administration to a mammal such as a human).

This invention also provides a method of method of ameliorating asymptom of atherosclerosis, or other pathology characterized by aninflammatory response in a mammal. The method involves administering tothe mammal (e.g. a human) a polypeptide or a concatamer of a polypeptidecomprising an amphipathic helical polypeptide having charged residues onthe polar face of the polypeptide and possessing a wide non-polar faceas described herein. In certain embodiments, the mammal is a human (e.g.a human diagnosed as having or as being at risk for atherosclerosis,stroke, or other pathology associated with an inflammatory response). Incertain embodiments, the mammal is non-human mammal (e.g. canine,feline, bovine, equine, porcine, etc.).

In another embodiment, this invention provides a method of amelioratinga symptom of a pathology characterized by an inflammatory response (e.g.a symptom of rheumatoid arthritis, lupus erythematous, polyarteritisnodosa, osteoporosis, Altzheimer's disease and viral illnesses such asinfluenza A, etc). The method involves administering to the mammal (e.g.human) a polypeptide or a concatamer of a polypeptide comprising anamphipathic helical polypeptide having charged residues on the polarface of the polypeptide and possessing a wide non-polar face asdescribed herein.

This invention also provides a kit for ameliorating a symptom ofatherosclerosis or another pathology characterized by an inflammatoryresponse. The kit typically includes a container containing one or moreof the polypeptides described herein. The polypeptide(s) can be combinedwith a pharmaceutically acceptable excipient (e.g. in a unit dosageformulation for oral, nasal, rectal, injectible administration). The kitcan additionally include instructional materials teaching the use of thepolypeptide for ameliorating one or more symptoms of atherosclerosis orof a pathology characterized by an inflammatory response.

In still another embodiment, this invention provides a method ofmitigating or preventing a coronary complication associated with anacute phase response to an inflammation in a mammal, wherein saidcoronary complication is a symptom of atherosclerosis. The methodinvolves administering to a mammal having the acute phase response, orat risk for the acute phase response, one or more polypeptides describedherein. The administration can be by a route such as oraladministration, nasal administration, rectal administration,intraperitoneal injection, and intravascular injection, subcutaneousinjection, transcutaneous administration, intramuscular injection, andthe like. In certain embodiments, the polypeptide is administered incombination with an all L-form of the same polypeptide. In certainembodiments, the polypeptide(s) are provided as a unit formulation in apharmaceutically acceptable excipient. The acute phase response can bean inflammatory response associated with a recurrent inflammatorydisease. In certain embodiments, the acute phase response is associatedwith a disease including, but not limited to leprosy, tuberculosis,systemic lupus erythematosus, polymyalgia rheumatica, polyarteritisnodosa, scleroderma, idiopathic pulmonary fibrosis, chronic obstructivepulmonary disease, Alzheimers Disease and AIDS, polymyalgia rheumatica,polyarteritis nodosa, scleroderma, idiopathic pulmonary fibrosis,chronic obstructive pulmonary disease, Alzheimers Disease, AIDS,coronary calcification, calcific aortic stenosis, osteoporosis, andrheumatoid arthritis. In certain embodiments, the acute phase responseis an inflammatory response associated with a condition such as abacterial infection, a viral infection, a fungal infection, an organtransplant, a wound, an implanted prosthesis, parasitic infection,sepsis, endotoxic shock syndrome, and biofilm formation.

This invention also provides a method of mitigating or preventing acoronary complication associated with an acute phase response to aninflammation in a mammal where the coronary complication is a symptom ofatherosclerosis. The method involves assaying the mammal (e.g. a human)for an acute phase protein (APP) level indicative of an acute phaseresponse or a significant risk of an acute phase response; andadministering to a mammal showing an acute phase protein (APP) levelindicative of an acute phase response a polypeptide as described herein.The acute phase protein (APP) can be a positive APR such as serumamyloid A, c-reactive protein, serum amyloid P component, C2 complementprotein, C3 complement protein, C4 complement protein, C5 complementprotein, C9 complement protein, B complement protein, C1 inhibitor, C4binding protein, fibrinogen, von Willebrand factor, α1-antitrypsin,α1-antichymotrypsin, α2 antiplasmin, heparin cofactor II, plasminogenactivator inhibitor I, haptoglobin, haemopexin, ceruloplasmin, manganesesuperoxide dismutase, (α1-acid glycoprotein, haeme oxygenase, mannosebinding protein, leukocyte protein I, lipoprotein (a), andlipopolysaccharide binding protein and/or a negative APR such asconsisting of albumin, prealbumin, transferin, apoAI, apoAII, α2-HSglycoprotein, inter-α-trypsin inhibitor, histidine rich glycoprotein.

Definitions.

The terms “isolated”, “purified”, or “biologically pure” when referringto an isolated polypeptide refer to material that is substantially oressentially free from components that normally accompany it as found inits native state. With respect to nucleic acids and/or polypeptides theterm can refer to nucleic acids or polypeptides that are no longerflanked by the sequences typically flanking them in nature. Chemicallysynthesized polypeptides are “isolated” because they are not found in anative state (e.g. in blood, serum, etc.). In certain embodiments, theterm “isolated” indicates that the polypeptide is not found in nature.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidues is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

The term “an amphipathic helical peptide” refers to a peptide comprisingat least one amphipathic helix (amphipathic helical domain). Certainamphipathic helical peptides of this invention can comprise two or more(e.g. 3, 4, 5, etc.) amphipathic helices.

The term “class A amphipathic helix” refers to a protein structure thatforms an α-helix producing a segregation of a polar and nonpolar faceswith the positively charged residues residing at the polar-nonpolarinterface and the negatively charged residues residing at the center ofthe polar face (see, e.g., “Segrest et al. (1990) Proteins: Structure,Function, and Genetics 8: 103-117).

“Apolipoprotein J” (apo J) is known by a variety of names includingclusterin, TRPM2, GP80, and SP 40,40 (Fritz (1995) Pp 112 In: Clusterin:Role in Vertebrate Development, Function, and Adaptation (Harmony JAKEd.), R. G. Landes, Georgetown, Tex.,). It was first described as aheterodimeric glycoprotein and a component of the secreted proteins ofcultured rat Sertoli cells (Kissinger et al. (1982) Biol Reprod;27:233240). The translated product is a single-chain precursor proteinthat undergoes intracellular cleavage into a disulfide-linked 34 kDaαsubunit and a 47 kDa βsubunit Collard and Griswold (187) Biochem., 26:3297-3303). It has been associated with cellular injury, lipidtransport, apoptosis and it may be involved in clearance of cellulardebris caused by cell injury or death. Clusterin has been shown to bindto a variety of molecules with high affinity including lipids, peptides,and proteins and the hydrophobic probe 1-anilino-8-naphthalenesulfonate(Bailey et al. (2001) Biochem., 40: 11828-11840).

The class G amphipathic helix is found in globular proteins, and thus,the name class G. The feature of this class of amphipathic helix is thatit possesses a random distribution of positively charged and negativelycharged residues on the polar face with a narrow nonpolar face. Becauseof the narrow nonpolar face this class does not readily associate withphospholipid (see, Segrest et al. (1990) Proteins: Structure, Function,and Genetics. 8: 103-117; also see Erratum (1991) Proteins: Structure,Function and Genetics, 9: 79). Several exchangeable apolipoproteinspossess similar but not identical characteristics to the G amphipathichelix. Similar to the class G amphipathic helix, this other classpossesses a random distribution of positively and negatively chargedresidues on the polar face. However, in contrast to the class Gamphipathic helix which has a narrow nonpolar face, this class has awide nonpolar face that allows this class to readily bind phospholipidand the class is termed G* to differentiate it from the G class ofamphipathic helix (see Segrest et al. (1992) J. Lipid Res., 33: 141-166;also see Anantharamaiah et al. (1993) Pp. 109-142 In: The AmphipathicHelix, Epand, R. M. Ed CRC Press, Boca Raton, Fla.). Computer programsto identify and classify amphipathic helical domains have been describedby Jones et al.(1992) J. Lipid Res. 33: 287-296) and include, but arenot limited to the helical wheel program (WHEEL or WHEEL/SNORKEL),helical net program (HELNET, HELNET/SNORKEL, HELNET/Angle), program foraddition of helical wheels (COMBO or COMBO/SNORKEL), program foraddition of helical nets (COMNET, COMNET/SNORKEL, COMBO/SELECT,COMBO/NET), consensus wheel program (CONSENSUS, CONSENSUS/SNORKEL), andthe like.

The term “ameliorating” when used with respect to “ameliorating one ormore symptoms of atherosclerosis” refers to a reduction, prevention, orelimination of one or more symptoms characteristic of atherosclerosisand/or associated pathologies. Such a reduction includes, but is notlimited to a reduction or elimination of oxidized phospholipids, areduction in atherosclerotic plaque formation and rupture, a reductionin clinical events such as heart attack, angina, or stroke, a decreasein hypertension, a decrease in inflammatory protein biosynthesis,reduction in plasma cholesterol, and the like.

The term “enantiomeric amino acids” refers to amino acids that can existin at least two forms that are nonsuperimposable mirror images of eachother. Most amino acids (except glycine) are enantiomeric and exist in aso-called L-form (L amino acid) or D-form (D amino acid). Most naturallyoccurring amino acids are “L” amino acids. The terms “D amino acid” and“L amino acid” are used to refer to absolute configuration of the aminoacid, rather than a particular direction of rotation of plane-polarizedlight. The usage herein is consistent with standard usage by those ofskill in the art.

The term “protecting group” refers to a chemical group that, whenattached to a functional group in an amino acid (e.g. a side chain, analpha amino group, an alpha carboxyl group, etc.) blocks or masks theproperties of that functional group. Preferred amino-terminal protectinggroups include, but are not limited to acetyl, or amino groups. Otheramino-terminal protecting groups include, but are not limited to alkylchains as in fatty acids, propeonyl, formyl and others. Preferredcarboxyl terminal protecting groups include, but are not limited togroups that form amides or esters.

The phrase “protect a phospholipid from oxidation by an oxidizing agent”refers to the ability of a compound to reduce the rate of oxidation of aphospholipid (or the amount of oxidized phospholipid produced) when thatphospholipid is contacted with an oxidizing agent (e.g. hydrogenperoxide, 13-(S)-HPODE, 15-(S)-HPETE, HPODE, HPETE, HODE, HETE, etc.).

The terms “low density lipoprotein” or “LDL” is defined in accordancewith common usage of those of skill in the art. Generally, LDL refers tothe lipid-protein complex which when isolated by ultracentrifugation isfound in the density range d=1.019 to d=1.063.

The terms “high density lipoprotein” or “HDL” is defined in accordancewith common usage of those of skill in the art. Generally “HDL” refersto a lipid-protein complex which when isolated by ultracentrifugation isfound in the density range of d=1.063 to d=1.21.

The term “Group I HDL” refers to a high density lipoprotein orcomponents thereof (e.g. apo A-I, paraoxonase, platelet activatingfactor acetylhydrolase, etc.) that reduce oxidized lipids (e.g. in lowdensity lipoproteins) or that protect oxidized lipids from oxidation byoxidizing agents.

The term “Group II HDL” refers to an HDL that offers reduced activity orno activity in protecting lipids from oxidation or in repairing (e.g.reducing) oxidized lipids.

The term “HDL component” refers to a component (e.g. molecules) thatcomprises a high density lipoprotein (HDL). Assays for HDL that protectlipids from oxidation or that repair (e.g. reduce oxidized lipids) alsoinclude assays for components of HDL (e.g. apo A-I, paraoxonase,platelet activating factor acetylhydrolase, etc.) that display suchactivity.

The term “human apo A-I peptide” refers to a full-length human apo A-Ipeptide or to a fragment or domain thereof comprising a class Aamphipathic helix.

A “monocytic reaction” as used herein refers to monocyte activitycharacteristic of the “inflammatory response” associated withatherosclerotic plaque formation. The monocytic reaction ischaracterized by monocyte adhesion to cells of the vascular wall (e.g.cells of the vascular endothelium), and/or chemotaxis into thesubendothelial space, and/or differentiation of monocytes intomacrophages.

The term “absence of change” when referring to the amount of oxidizedphospholipid refers to the lack of a detectable change, more preferablythe lack of a statistically significant change (e.g. at least at the85%, preferably at least at the 90%, more preferably at least at the95%, and most preferably at least at the 98% or 99% confidence level).The absence of a detectable change can also refer to assays in whichoxidized phospholipid level changes, but not as much as in the absenceof the protein(s) described herein or with reference to other positiveor negative controls.

The following abbreviations are used herein: PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; POVPC:1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine; PGPC:1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine; PEIPC:1-palmitoyl-2-(5,6-epoxyisoprostane E₂)-sn-glycero-3-phosphocholine;ChC18:2: cholesteryl linoleate; ChC18: 2-OOH: cholesteryl linoleatehydroperoxide; DMPC: 1,2-ditetradecanoyl-rac-glycerol-3-phosphocholine;PON: paraoxonase; HPF: Standardized high power field; PAPC:L-α-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; BL/6:C57BL/6J; C3H: C3H/HeJ.

The term “conservative substitution” is used in reference to proteins orpeptides to reflect amino acid substitutions that do not substantiallyalter the activity (specificity (e.g. for lipoproteins))or bindingaffinity (e.g. for lipids or lipoproteins)) of the molecule. Typicallyconservative amino acid substitutions involve substitution one aminoacid for another amino acid with similar chemical properties (e.g.charge or hydrophobicity). The following six groups each contain aminoacids that are typical conservative substitutions for one another: 1)Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.With respect to the peptides of this invention sequence identity isdetermined over the full length of the peptide.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng & Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins & Sharp (1989) CABIOS 5: 151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993) Proc. Natl. Acad.Sci. USA, 90: 5873-5787). One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the effect of D4F (Navab, et al. (2002)Circulation, 105: 290-292) and apoJ peptide 336 made from D amino acids(D-J336*) on the prevention of LDL-induced monocyte chemotactic activityin vitro in a co-incubation experiment. The data are mean±SD of thenumber of migrated monocytes in nine high power fields in quadruplecultures. (D-J336=Ac-LLEQLNEQFNWVSRLANLTEGE-NH₂, SEQ ID NO:1).

FIG. 2 illustrates the prevention of LDL-induced monocyte chemotacticactivity by pre-treatment of artery wall cells with D-J336 as comparedto D-4F. The data are mean±SD of the number of migrated monocytes innine high power fields in quadruple cultures.

FIG. 3 illustrates he effect of apo J peptide mimetics on HDL protectivecapacity in LDL receptor null mice. The values are the mean±SD of thenumber of migrated monocytes in 9 high power fields from each ofquadruple assay wells.

FIG. 4 illustrates protection against LDL-induced monocyte chemotacticactivity by HDL from apo E null mice given oral peptides. The values arethe mean±SD of the number of migrated monocytes in 9 high power fieldsfrom each of quadruple assay wells. Asterisks indicate significantdifference (p<0.05) as compared to No Peptide mHDL.

FIG. 5 illustrates the effect of oral apo A-I peptide mimetic and apojpeptide on LDL susceptibility to oxidation. The values are the mean±SDof the number of migrated monocytes in 9 high power fields from each ofquadruple assay wells. Asterisks indicate significant difference(p<0.05) as compared to No Peptide LDL.

FIG. 6 illustrates the effect of oral apoA-1 peptide mimetic and apoJpeptide on HDL protective capacity. The values are the mean±SD of thenumber of migrated monocytes in 9 high power fields from each ofquadruple assay wells. Asterisks indicate significant difference(p<0.05) as compared to No Peptide mHDL.

FIG. 7 illustrates the Effect of oral apoA-1 peptide mimetic and apojpeptide on plasma paraoxonase activity. The values are the mean±SD ofreadings from quadruple plasma aliquots. Asterisks indicate significantdifferences (p<0.05) as compared to No Peptide control plasma.

DETAILED DESCRIPTION

I. Mitigation of a Symptom of Atherosclerosis.

This invention pertains to the surprising discovery that amphipathichelical peptide analogues possessing distributed (e.g. randomlydistributed, haphazardly distributed, etc.) charged residues on thepolar face of the peptide possess anti-inflammatory properties and arecapable of mediating a symptom of atherosclerosis or other pathologycharacterized by an inflammatory response (e.g., rheumatoid arthritis,lupus erythematous, polyarteritis nodosa, and osteoporosis). Preferredpeptides of this invention generally mimic the amphipathic helicaldomain(s) of apolipoprotein J (apo J).

In certain preferred embodiments, the peptides are amphipathic helicalpeptide analogues possessing distributed charged residues (positivelyand/or negatively charged residues) on the polar face of the peptide andpossessing a wide nonpolar face (termed a globular protein like, G*)amphipathic helical domain. Such amphipathic helical G* domains arecharacteristic of apo J and certain other apoproteins (e.g. apo AI, apoAIV, apo E, apo CII, apo CIII, and the like, but not apo A-II or apoC-I). The peptides of this invention preferably range from about 10 toabout 100 amino acids in length, more preferably from about 10 to about60 or 80 amino acids in length, and most preferably from about 10, 15,or 20 amino acids to about 40 or 50 amino acids in length. In certainembodiments, the peptides range from about 10 to about 40 amino acids inlength. Certain particularly preferred peptides of this invention showgreater than about 40%, preferably greater than about 50% or 60%, morepreferably greater than about 70% or 80% and most preferably greaterthan about 90% or 95% sequence identity with apo J or fragments thereof(ranging in length from about 10 to about 40 amino acids, e.g. over thesame length as the peptide in question).

It was a surprising discovery of this invention that such peptides,particularly when comprising one or more D-form amino acids retain thebiological activity of the corresponding L-form peptide. Moreover, thesepeptides show in vivo activity, even when delivered orally. The peptidesshow elevated serum half-life, and the ability to mitigate orprevent/inhibit one or more symptoms of atherosclerosis.

We discovered that normal HDL inhibits three steps in the formation ofmildly oxidized LDL. In those studies (see, e.g. WO 02/15923) wedemonstrated that treating human LDL in vitro with apo A-I or an apo A-Imimetic peptide (37 pA) removed seeding molecules from the LDL thatincluded HPODE and HPETE. These seeding molecules were required forcocultures of human artery wall cells to be able to oxidize LDL and forthe LDL to induce the artery wall cells to produce monocyte chemotacticactivity. We also demonstrated that after injection of apo A-I into miceor infusion into humans, the LDL isolated from the mice or humanvolunteers was resistant to oxidation by human artery wall cells and didnot induce monocyte chemotactic activity in the artery wall cellcocultures.

Without being bound to a particular theory, we believe the peptides ofthis invention function in a manner similar to the activity of the apoA-I mimetics described in PCT publication WO 02/15923. In particular, webelieve the present invention functions in part by increasing theant-inflammatory properties of HDL. In particular, we believe thepeptides of this invention bind seeding molecules in LDL that arenecessary for LDL oxidation and then carry the seeding molecules awaywhere there are ultimately excreted.

We have discovered that peptides that mimic the amphipathic helicaldomain(s) of apolipoprotein J are particularly effective in protectingLDL against oxidation by arterial wall cells and in reducing LDL-inducedmonocyte chemotactic activity that results from the oxidation of LDL byhuman artery wall cells. Apo J possesses a wide nonpolar face termedglobular proteinlike, or G* amphipathic helical domains. The class Gamphipathic helix is found in globular proteins, and thus, the nameclass G. The feature of this class of amphipathic helix is that itpossesses a random/haphazard distribution of positively charged andnegatively charged residues on the polar face with a narrow nonpolarface. Because of the narrow nonpolar face this class does not readilyassociate with phospholipid (see Segrest et al. (1990) Proteins:Structure, Function, and Genetics. 8: 103-117; also see Erratum (1991)Proteins: Structure, Function and Genetics, 9: 79). Several exchangeableapolipoproteins possess similar but not identical characteristics to theG amphipathic helix. Similar to the class G amphipathic helix, thisother class possesses a random distribution of positively and negativelycharged residues on the polar face. However, in contrast to the class Gamphipathic helix which has a narrow nonpolar face, this class has awide nonpolar face that allows this class to readily bind phospholipidand the class is termed G* to differentiate it from the G class ofamphipathic helix (see Segrest et al. (1992) J. Lipid Res., 33: 141-166;also see Anantharamaiah et al. (1993) Pp. 109-142 In The AmphipathicHelix, Epand, R. M. Ed., CRC Press, Boca Raton, Fla.).

It was a surprising discovery of this invention that the amphipathichelical peptides of this invention required to render human artery wallcells incapable of oxidizing LDL was substantially less than thatrequired for apo AI mimetic peptides such as D4F in a preincubation withartery wall cells.

We have demonstrated that oral administration of an apo AI mimeticpeptide synthesized from D amino acids dramatically reducesatherosclerosis in mice independent of changes in plasma or HDLcholesterol concentrations. Similar to the action of the apo A-Imimetics, we believe that synthetic peptides mimicking the amphipathichelical domains of apo J that are synthesized from D amino acids can begiven orally or by other routes including injection and will ameliorateatherosclerosis and other chronic inflammatory conditions.

The peptides of this invention can comprise all L-form amino acids.However, the inventors believe peptides comprising one or more D-formamino acids and preferably all D-form amino acids (all enantiomericamino acids are D form) provide for more effective delivery via oraladministration and will be more stable in the circulation. Particularlypreferred peptides are blocked at one or both termini (e.g. with theN-terminus acetylated and the C-terminus amidated).

The protective function of the peptides of this invention is illustratedin Example 1. The in vitro concentration of the new class of peptidesnecessary to prevent LDL-induced monocyte chemotactic activity by humanartery wall cells is 10 to 25 times less than the concentration requiredfor an apoA-I mimetic (D4F) (compare DJ336 to D4F in FIG. 1). Similarly,in a preincubation the peptides of this invention were 1025 times morepotent in preventing LDL oxidation by artery wall cells (compare DJ336to D4F in FIG. 2). As shown in FIG. 3, when DJ335 was given orally toLDL receptor null mice it was essentially as effective as D4F inrendering HDL more protective in preventing LDL-induced monocytechemotactic activity.

FIG. 4 demonstrates that when added to the drinking water a peptide ofthis invention (DJ336) was as potent as D4F in enhancing HDL protectivecapacity in apo E null mice. FIG. 5 demonstrates that, when added to thedrinking water, a peptide of this invention DJ336 was slightly morepotent than D4F in rendering the LDL from apo E null mice resistant tooxidation by human artery wall cells as determined by the induction ofmonocyte chemotactic activity. FIG. 6 demonstrates that when added tothe drinking water DJ336 was as potent as D4F in causing HDL to inhibitthe oxidation of a phospholipid PAPC by the oxidant HPODE in a humanartery wall coculture as measured by the generation of monocytechemotactic activity (see Navab et al. (2001) J. Lipid Res. 42:1308-1317 for an explanation of the test system). FIG. 7 demonstratesthat, when added to the drinking water, DJ336 was at least as potent asD4F in increasing the paraoxonase activity of apo E null mice.

Since many inflammatory conditions have been suspected to be mediated atleast in part by oxidized lipids, we believe that this invention is alsoeffective in ameliorating conditions that are known or suspected to bedue to the formation of oxidized lipids. These include, but are notlimited to, rheumatoid arthritis, lupus erythematous, polyarteritisnodosa, and osteoporosis.

Without being bound to a particular theory, we believe administration(e.g. injection) of one or more of the peptides of this invention willameliorate the signs and symptoms of influenza A. In addition, thepeptide will dramatically reduced the influx of macrophages into theartery wall. This will have great utility in reducing the high rate ofheart attack and stroke after influenza and other viral infections.Thus, the peptides of this invention can be used to ameliorate the signsand symptoms of influenza and various other viral illnesses and reducethe incidence of heart attack and stroke that often follows these viralillnesses.

In view of the foregoing, in one embodiment, this invention providesmethods for ameliorating and/or preventing one or more symptoms ofatherosclerosis and/or a pathology associated with (characterized by) aninflammatory response. The methods typically involve administering to anorganism, preferably a mammal, more preferably a human one or more ofthe peptides of this invention (or mimetics of such peptides). Thepeptide(s) can be administered, as described herein, according to any ofa number of standard methods including, but not limited to injection,suppository, nasal spray, time-release implant, transdermal patch, andthe like. In one particularly preferred embodiment, the peptide(s) areadministered orally (e.g. as a syrup, capsule, or tablet).

The methods involve the administration of a single polypeptide of thisinvention or the administration of two or more different polypeptides.The polypeptides can be provided as monomers or in dimeric, oligomericor polymeric forms. In certain embodiments, the multimeric forms maycomprise associated monomers (e.g. ionically or hydrophobically linked)while certain other multimeric forms comprise covalently linked monomers(directly linked or through a linker).

While the invention is described with respect to use in humans, it isalso suitable for animal, e.g. veterinary use. Thus preferred organismsinclude, but are not limited to humans, non-human primates, canines,equines, felines, porcines, ungulates, largomorphs, and the like.

The methods of this invention are not limited to humans or non-humananimals showing one or more symptom(s) of atherosclerosis (e.g.hypertension, plaque formation and rupture, reduction in clinical eventssuch as heart attack, angina, or stroke, high levels of plasmacholesterol, high levels of low density lipoprotein, high levels of verylow density lipoprotein, or inflammatory proteins, etc.), but are usefulin a prophylactic context. Thus, the peptides of this invention (ormimetics thereof) may be administered to organisms to prevent theonset/development of one or more symptoms of atherosclerosis.Particularly preferred subjects in this context are subjects showing oneor more risk factors for atherosclerosis (e.g. family history,hypertension, obesity, high alcohol consumption, smoking, high bloodcholesterol, high blood triglycerides, elevated blood LDL, VLDL, IDL, orlow HDL, diabetes, or a family history of diabetes, high blood lipids,heart attack, angina or stroke, etc.).

In addition to methods of use of the atherosclerosis-inhibiting peptidesof this invention, this invention also provides the peptides themselves,the peptides formulated as pharmaceuticals, particularly for oraldelivery, and kits for the treatment and/or prevention of one or moresymptoms of atherosclerosis.

II. Mitigation of a Symptom of Atherosclerosis Associated with an AcuteInflammatory Response.

The atherosclerosis-inhibiting peptides of this invention are alsouseful in a number of other contexts. In particular, we have observedthat cardiovascular complications (e.g. atherosclerosis, stroke, etc.)frequently accompany or follow the onset of an acute phase inflammatoryresponse. Such an acute state inflammatory response is often associatedwith a recurrent inflammatory disease (e.g., leprosy, tuberculosis,systemic lupus erythematosus, and rheumatoid arthritis), a viralinfection (e.g. influenza), a bacterial infection, a fungal infection,an organ transplant, a wound or other trauma, an implanted prosthesis, abiofilm, and the like.

It was a surprising discovery of this invention that administration ofone or more of the peptides described herein, can reduce or prevent theformation of oxidized phospholipids during or following an acute phaseresponse and thereby mitigate or eliminate cardiovascular complicationsassociated with such a condition.

Thus, for example, we have demonstrated that a consequence of influenzainfection is the diminution in paraoxonase and platelet activatingacetylhydrolase activity in the HDL. Without being bound by a particulartheory, we believe that, as a result of the loss of these HDL enzymaticactivities and also as a result of the association of pro-oxidantproteins with HDL during the acute phase response, HDL is no longer ableto prevent LDL oxidation and was no longer able to prevent theLDL-induced production of monocyte chemotactic activity by endothelialcells.

We observed that in a subject injected with very low dosages of apo-AImimetics (e.g. 20 micrograms for mice) daily after infection with theinfluenza A virus paraoxonase levels did not fall and the biologicallyactive oxidized phospholipids were not generated beyond background (see,e.g., WO 02/15923, PCT/US01/26497).

It was surprising discovery that the class of peptides described hereincan act in manner similar to the apo-I mimetics described in WO02/15923. In view of this discovery, it is believed that the peptides ofthis invention can be administered (e.g. orally or by injection) topatients with known coronary artery disease during influenza infectionor other events that can generate an acute phase inflammatory response(e.g. due to viral infection, bacterial infection, trauma, transplant,various autoimmune conditions, etc.) and thus we can prevent by thisshort term treatment the increased incidence of heart attack and strokeassociated with pathologies that generate such inflammatory states.

Thus, in certain embodiments, this invention contemplates administeringone or more of the peptides of this invention to a subject at risk for,or incurring, an acute inflammatory response and/or at risk for orincurring a symptom of atherosclerosis.

For example, a person having or at risk for coronary disease mayprophylactically be administered a polypeptide of this invention duringflu season. A person (or animal) subject to a recurrent inflammatorycondition, e.g. rheumatoid arthritis, various autoimmune diseases, etc.,can be treated with a polypeptide of this invention to mitigate orprevent the development of atherosclerosis or stroke. A person (oranimal) subject to trauma, e.g. acute injury, tissue transplant, etc.can be treated with a polypeptide of this invention to mitigate thedevelopment of atherosclerosis or stroke.

In certain instances such methods will entail a diagnosis of theoccurrence or risk of an acute inflammatory response. The acuteinflammatory response typically involves alterations in metabolism andgene regulation in the liver. It is a dynamic homeostatic process thatinvolves all of the major systems of the body, in addition to theimmune, cardiovascular and central nervous system. Normally, the acutephase response lasts only a few days; however, in cases of chronic orrecurring inflammation, an aberrant continuation of some aspects of theacute phase response may contribute to the underlying tissue damage thataccompanies the disease, and may also lead to further complications, forexample cardiovascular diseases or protein deposition diseases such asamyloidosis.

An important aspect of the acute phase response is the radically alteredbiosynthetic profile of the liver. Under normal circumstances, the liversynthesizes a characteristic range of plasma proteins at steady stateconcentrations. Many of these proteins have important functions andhigher plasma levels of these acute phase reactants (APRs) or acutephase proteins (APPs) are required during the acute phase responsefollowing an inflammatory stimulus. Although most APRs are synthesizedby hepatocytes, some are produced by other cell types, includingmonocytes, endothelial cells, fibroblasts and adipocytes. Most APRs areinduced between 50% and several-fold over normal levels. In contrast,the major APRs can increase to 1000-fold over normal levels. This groupincludes serum amyloid A (SAA) and either C-reactive protein (CRP) inhumans or its homologue in mice, serum amyloid P component (SAP).So-called negative APRs are decreased in plasma concentration during theacute phase response to allow an increase in the capacity of the liverto synthesize the induced APRs.

In certain embodiments, the acute phase response, or risk therefore isevaluated by measuring one or more APPs. Measuring such markers is wellknown to those of skill in the art, and commercial companies exist thatprovide such measurement (e.g. AGP measured by Cardiotech Services,Louisville, Ky.).

III. Mitigation of a Symptom or Condition Associated with CoronaryCalcification and Osteoporosis.

We have also identified oxidized lipids as a cause of coronarycalcification and osteoporosis. Moreover, without being bound to aparticularly theory, we believe the same mechanisms are involved in thepathogenesis of calcific aortic stenosis.

Thus, in certain embodiments, this invention contemplates the use of thepeptides described herein to inhibit or prevent a symptom of a diseasesuch as polymyalgia rheumatica, polyarteritis nodosa, scleroderma,idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease,Alzheimers Disease, AIDS, coronary calcification, calcific aorticstenosis, osteoporosis, and the like.

IV. Preferred Peptides and their Preparation.

Preferred Peptides.

It was a discovery of this invention that peptides that mimicking theamphipathic helical domains of apo J are capable of mitigating one ormore symptoms of atherosclerosis and/or other pathologies characterizedby an inflammatory response. Apolipoprotein J possesses a wide nonpolarface termed globular protein-like, or G* amphipathic helical domains.The class G amphipathic helix is found in globular proteins, and thus,the name class G. This class of amphipathic helix is characterized by arandom distribution of positively charged and negatively chargedresidues on the polar face with a narrow nonpolar face. Because of thenarrow nonpolar face this class does not readily associate withphospholipids. The G* of amphipathic helix possesses similar, but notidentical, characteristics to the G amphipathic helix. Similar to theclass G amphipathic helix, the G* class peptides possesses a randomdistribution of positively and negatively charged residues on the polarface. However, in contrast to the class G amphipathic helix which has anarrow nonpolar face, this class has a wide nonpolar face that allowsthis class to readily bind phospholipid and the class is termed G* todifferentiate it from the G class of amphipathic helix.

A variety of suitable peptides of this invention that are related to G*amphipathic helical domains of apo J are illustrated in Table 1.

TABLE 1 Preferred peptides for use in this invention related to g*amphipathic helical domains of apo J. Amino Acid Sequence SEQ ID NOLLEQLNEQFNWVSRLANLTEGE 1 LLEQLNEQFNWVSRLANL 2 NELQEMSNQGSKYVNKEIQNAVNGV3 IQNAVNGVKQIKTLIEKTNEE 4 RKTLLSNLEEAKKKKEDALNETRESETKLKEL 5PGVCNETMMALWEECK 6 PCLKQTCMKFYARVCR 7 ECKPCLKQTCMKFYARVCR 8 LVGRQLEEFL 9MNGDRIDSLLEN 10 QQTHMLDVMQD 11 FSRASSIIDELFQD 12 PFLEMTHEAQQAMDI 13PTEFTREGDDD 14 RMKDQCDKCREILSV 15 PSQAKLRRELDESLQVAERLTRKYNELLKSYQ 16LLEQLNEQFNWVSRLANLTQGE 17 DQYYLRVTTVA 18 PSGVTEVVVKLFDS 19PKFMETVAEKALQEYRKKHRE 20

The peptides of this invention, however, are not limited to G* variantsof apo J. Generally speaking G* domains from essentially any otherprotein preferably apo proteins are also suitable. The particularsuitability of such proteins can readily be determined using assays forprotective activity (e.g. protecting LDL from oxidation, and the like),e.g. as illustrated herein in the Examples. Some particularly preferredproteins include G* amphipathic helical domains or variants thereof(e.g. conservative substitutions, and the like) of proteins including,but not limited to apo AI, apo AIV, apo E, apo CII, apo CIII, and thelike.

Certain preferred peptides for related to G* amphipathic helical domainsrelated to apoproteins other than apo J are illustrated in Table 2.

TABLE 2 Peptides for use in this invention related to G* amphipathichelical domains related to apoproteins other than apo J. SEQ ID AminoAcid Sequence NO WDRVKDLATVYVDVLKDSGRDYVSQF 21 (Related to the 8 to 33region of apo AI) VATVMWDYFSQLSNNAKEAVEHLQK 22 (Related to the 7 to 31region of apo AIV) RWELALGRFWDYLRWVQTLSEQVQEEL 23 (Related to the 25 to51 region of apo E) LSSQVTQELRALMDETMKELKELKAYKSELEEQLT 24 (Related tothe 52 to 83 region of apo E) ARLSKELQAAQARLGADMEDVCGRLV 25 (Related tothe 91 to 116 region of apo E) VRLASHLRKLRKRLLRDADDLQKRLA 26 (Related tothe 135 to 160 region of apo E) PLVEDMQRQWAGLVEKVQA 27 (267 to 285 ofapo E.27) MSTYTGIFTDQVLSVLK 28 (Related to the 60 to 76 region of apoCII) LLSFMQGYMKHATKTAKDALSS 29 (Related to the 8 to 29 region of apoCIII)

While the various peptides listed in Table 1 and Table 2 are shown withno protecting groups, in certain embodiments (e.g. particularly for oraladministration), they bear one or two protecting groups, more preferablyterminal protecting groups. Thus, for example, in certain embodiments,any of the peptides descry bed herein can bear, e.g. an acetyl groupprotecting the amino terminus and/or an amide group protecting thecarboxyl terminus. One example of such a “dual protected peptide isAc-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-NH₂ (SEQ ID NO:1 withblocking groups), either or both of these protecting groups can beeliminated and/or substituted with another protecting group as describedherein. In particularly preferred embodiments, the peptides comprise oneor more D-form (dextro rather than levo) amino acids as describedherein. In certain embodiments at least two enantiomeric amino acids,more preferably at least 4 enantiomeric amino acids and most preferablyat least 8 or 10 enantiomeric amino acids are “D” form amino acids. Incertain embodiments every amino acid (e.g. every enantiomeric aminoacid) of the peptides described herein is a D-form amino acid.

It is also noted that the peptides listed in Tables 1 and 2 are notfully inclusive. Using the teaching provided herein, other suitablepeptides can routinely be produced (e.g. by conservative orsemi-conservative substitutions (e.g. D replaced by E), extensions,deletions, and the like). Thus, for example, one embodiment utilizestruncations of any one or more of peptides identified by SEQ ID Nos:1-29.

Longer peptides are also suitable. Such longer peptides may entirelyform a class G or G* amphipathic helix, or the G amphipathic helix(helices) can form one or more domains of the peptide. In addition, thisinvention contemplates multimeric versions of the peptides. Thus, forexample, the peptides illustrated in Tables 1 or 2 can be coupledtogether (directly or through a linker (e.g. a carbon linker, or one ormore amino acids) with one or more intervening amino acids). Suitablelinkers include, but are not limited to Proline (-Pro-), Gly₄Ser₃ (SEQID NO:30), and the like. Thus, one illustrative multimeric peptideaccording to this invention is (D-J336)-P-(D-J336) (i.e.Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-P-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-NH₂,SEQ ID NO:31).

This invention also contemplates the use of “hybrid” peptides comprisinga one or more G or G* amphipathic helical domains and one or more classA amphipathic helices. Suitable class A amphipathic helical peptides aredescribed in PCT publication WO 02/15923. Thus, by way of illustration,one such “hybrid” peptide is (D-J336)-Pro-(4F) (i.e.Ac-L-L-E-Q-L-N-E-Q-F-N-W-V-S-R-L-A-N-L-T-E-G-E-P-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH₂,SEQ ID NO:32), and the like. As indicated above, the peptides of thisinvention preferably comprise one or more D amino acids, more preferablywith every amino acid a D amino acid as described herein and/or havingone or both termini protected. Preferably at least 50% of theenantiomeric amino acids are “D” form, more preferably at least 80% ofthe enantiomeric amino acids are “D” form, and most preferably at least90% or even all of the enantiomeric amino acids are “D” form aminoacids.

It was a surprising discovery of this invention that, when theamphipathic helical peptides of this invention (e.g. as illustrated inFIGS. 3, 4, 5, 6, and 7) incorporated D amino acids they retained theiractivity even when administered orally. Moreover this oraladministration resulted in relatively efficient uptake and significantserum half-life thereby providing an efficacious method of mitigatingone or more symptoms of atherosclerosis and/or other conditionscharacterized by an inflammatory response.

Using the teaching provided herein, one of skill can routinely modifythe illustrated amphipathic helical peptides to produce other suitableapo J variants and/or amphipathic G helical peptides of this invention.For example, routine conservative or semi-conservative substitutions(e.g. E for D) can be made of the existing amino acids. The effect ofvarious substitutions on lipid affinity of the resulting peptide can bepredicted using the computational method described by Palgunachari etal. (1996) Arteriosclerosis, Thrombosis, & Vascular Biology 16: 328-338.The peptides can be lengthened or shortened as long as the class helixstructure(s) are preserved. In addition, substitutions can be made torender the resulting peptide more similar to peptide(s) endogenouslyproduced by the subject species.

New peptides can be designed and/or evaluated using computationalmethods. Computer programs to identify and classify amphipathic helicaldomains are well known to those of skill in the art and many have beendescribed by Jones et al.(1992) J. Lipid Res. 33: 287-296). Suchprograms include, but are not limited to the helical wheel program(WHEEL or WHEEL/SNORKEL), helical net program (HELNET, HELNET/SNORKEL,HELNET/Angle), program for addition of helical wheels (COMBO orCOMBO/SNORKEL), program for addition of helical nets (COMNET,COMNET/SNORKEL, COMBO/SELECT, COMBO/NET), consensus wheel program(CONSENSUS, CONSENSUS/SNORKEL), and the like.

While, in preferred embodiments, the peptides of this invention utilizenaturally-occurring amino acids or D forms of naturally occurring aminoacids, substitutions with non-naturally occurring amino acids (e.g.,methionine sulfoxide, methionine methylsulfonium, norleucine,episilon-aminocaproic acid, 4-aminobutanoic acid,tetrahydroisoquinoline-3-carboxylic acid, 8-aminocaprylic acid,4-aminobutyric acid, Lys(N(epsilon)-trifluoroacetyl), α-aminoisobutyricacid, and the like) are also contemplated.

In addition to the G* amphipathic helical peptides described herein,peptidomimetics are also contemplated herein. Peptide analogs arecommonly used in the pharmaceutical industry as non-peptide drugs withproperties analogous to those of the template peptide. These types ofnon-peptide compound are termed “peptide mimetics” or “peptidomimetics”(Fauchere (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINSp. 392; and Evans et al. (1987) J. Med. Chem. 30: 1229) and are usuallydeveloped with the aid of computerized molecular modeling. Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent therapeutic orprophylactic effect.

Generally, peptidomimetics are structurally similar to a paradigmpolypeptide (e.g. SEQ ID NO:1 shown in Table 1), but have one or morepeptide linkages optionally replaced by a linkage selected from thegroup consisting of: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂—, —CH₂SO—, etc by methods known in the artand further described in the following references: Spatola (1983) p. 267in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B.Weinstein, eds., Marcel Dekker, New York,; Spatola (1983) Vega Data 1(3)Peptide Backbone Modifications. (general review); Morley (1980) TrendsPharm Sci pp. 463-468 (general review); Hudson et al. (1979) Int J PeptProt Res 14:177-185 (—CH₂NH—, CH₂CH₂—); Spatola et al. (1986) Life Sci38: 1243-1249 (—CH₂—S); Hann, (1982) J Chem Soc Perkin Trans I 1307-314(—CH—CH—, cis and trans); Almquist et al. (1980) J Med Chem.23:1392-1398 (—COCH₂—); Jennings-White et al.(1982) Tetrahedron Lett.23:2533 (—COCH₂—); Szelke et al., European Appln. EP 45665 (1982) CA:97:39405 (1982) (—CH(OH)CH2-); Holladay et al. (1983) Tetrahedron Lett24:4401-4404 (—C(OH)CH₂—); and Hruby (1982) Life Sci., 31:189-199(—CH₂—S—)).

A particularly preferred non-peptide linkage is —CH₂NH—. Such peptidemimetics may have significant advantages over polypeptide embodiments,including, for example: more economical production, greater chemicalstability, enhanced pharmacological properties (half-life, absorption,potency, efficacy, etc.), reduced antigenicity, and others.

In addition, circularly permutations of the peptides described herein orconstrained peptides (including cyclized peptides) comprising aconsensus sequence or a substantially identical consensus sequencevariation may be generated by methods known in the art (Rizo andGierasch (1992) Ann. Rev. Biochem. 61: 387); for example, by addinginternal cysteine residues capable of forming intramolecular disulfidebridges which cyclize the peptide.

Peptide Preparation.

The peptides used in this invention can be chemically synthesized usingstandard chemical peptide synthesis techniques or, particularly wherethe peptide does not comprise “D” amino acid residues, can berecombinantly expressed. In certain embodiments, even peptidescomprising “D” amino acid residues are recombinantly expressed. Wherethe polypeptides are recombinantly expressed, a host organism (e.g.bacteria, plant, fungal cells, etc.) in cultured in an environment whereone or more of the amino acids is provided to the organism exclusivelyin a D form. Recombinantly expressed peptides in such a system thenincorporate those D amino acids.

In preferred embodiments the peptides are chemically synthesized by anyof a number of fluid or solid phase peptide synthesis techniques knownto those of skill in the art. Solid phase synthesis in which theC-terminal amino acid of the sequence is attached to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence is a preferred method for the chemical synthesis of thepolypeptides of this invention. Techniques for solid phase synthesis arewell known to those of skill in the art and are described, for example,by Barany and Merrifield (1963) Solid-Phase Peptide Synthesis; pp. 3-284in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methodsin Peptide Synthesis, Part A.; Merrifield et al. (1963) J. Am. Chem.Soc., 85: 2149-2156, and Stewart et al. (1984) Solid Phase PeptideSynthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.

In a most preferred embodiment, the peptides are synthesized by thesolid phase peptide synthesis procedure using a benzhyderylamine resin(Beckman Bioproducts, 0.59 mmol of NH₂/g of resin) as the solid support.The COOH terminal amino acid (e.g., t-butylcarbonyl-Phe) is attached tothe solid support through a 4-(oxymethyl)phenacetyl group. This is amore stable linkage than the conventional benzyl ester linkage, yet thefinished peptide can still be cleaved by hydrogenation. Transferhydrogenation using formic acid as the hydrogen donor is used for thispurpose. Detailed protocols used for peptide synthesis and analysis ofsynthesized peptides are described in a miniprint supplementaccompanying Anantharamaiah et al. (1985) J. Biol. Chem., 260(16):10248-10255.

It is noted that in the chemical synthesis of peptides, particularlypeptides comprising D amino acids, the synthesis usually produces anumber of truncated peptides in addition to the desired full-lengthproduct. The purification process (e.g. HPLC) typically results in theloss of a significant amount of the full-length product.

It was a discovery of this invention that, in the synthesis of a Dpeptide (e.g. D-4), in order to prevent loss in purifying the longestform one can dialyze and use the mixture and thereby eliminate the lastHPLC purification. Such a mixture loses about 50% of the potency of thehighly purified product (e.g. per wt of protein product), but themixture contains about 6 times more peptide and thus greater totalactivity.

D-form Amino Acids.

D-amino acids are incorporated at one or more positions in the peptidesimply by using a D-form derivatized amino acid residue in the chemicalsynthesis. D-form residues for solid phase peptide synthesis arecommercially available from a number of suppliers (see, e.g., AdvancedChem Tech, Louisville; Nova Biochem, San Diego; Sigma, St Louis; BachemCalifornia Inc., Torrance, etc.). The D-form amino acids can beincorporated at any position in the peptide as desired. Thus, forexample, in one embodiment, the peptide can comprise a single D-aminoacid, while in other embodiments, the peptide comprises at least two,generally at least three, more generally at least four, most generallyat least five, preferably at least six, more preferably at least sevenand most preferably at least eight D amino acids. In particularlypreferred embodiments, essentially every other (enantiomeric) amino acidis a D-form amino acid. In certain embodiments at least 90%, preferablyat least 90%, more preferably at least 95% of the enantiomeric aminoacids are D-form amino acids. In one particularly preferred embodiment,essentially every enantiomeric amino acid is a D-form amino acid.

Protecting Groups.

In certain embodiments, the one or more R-groups on the constituentamino acids and/or the terminal amino acids are blocked with aprotecting group. Without being bound by a particular theory, it was adiscovery of this invention that blockage, particularly of the aminoand/or carboxyl termini of the subject peptides of this inventiongreatly improves oral delivery and significantly increases serumhalf-life.

A wide number of protecting groups are suitable for this purpose. Suchgroups include, but are not limited to acetyl, amide, and alkyl groupswith acetyl and alkyl groups being particularly preferred for N-terminalprotection and amide groups being preferred for carboxyl terminalprotection. In certain particularly preferred embodiments, theprotecting groups include, but are not limited to alkyl chains as infatty acids, propeonyl, formyl, and others. Particularly preferredcarboxyl protecting groups include amides, esters, and ether-formingprotecting groups. In one preferred embodiment, an acetyl group is usedto protect the amino terminus and an amide group is used to protect thecarboxyl terminus. These blocking groups enhance the helix-formingtendencies of the peptides. Certain particularly preferred blockinggroups include alkyl groups of various lengths, e.g. groups having theformula: CH₃—(CH₂)_(n)—CO— where n ranges from about 1 to about 20,preferably from about 1 to about 16 or 18, more preferably from about 3to about 13, and most preferably from about 3 to about 10.

In certain particularly preferred embodiments, the protecting groupsinclude, but are not limited to alkyl chains as in fatty acids,propeonyl, formyl, and others. Particularly preferred carboxylprotecting groups include amides, esters, and ether-forming protectinggroups. In one preferred embodiment, an acetyl group is used to protectthe amino terminus and an amide group is used to protect the carboxylterminus. These blocking groups enhance the helix-forming tendencies ofthe peptides. Certain particularly preferred blocking groups includealkyl groups of various lengths, e.g. groups having the formula:CH₃—(CH₂)_(n)—CO— where n ranges from about 3 to about 20, preferablyfrom about 3 to about 16, more preferably from about 3 to about 13, andmost preferably from about 3 to about 10.

Other protecting groups include, but are not limited to Fmoc,t-butoxycarbonyl (t-BOC), 9-fluoreneacetyl group, 1-fluorenecarboxylicgroup, 9-florenecarboxylic group, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos),2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl),4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz),3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimentyl-2,6-diaxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy(cHxO),t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl(Ac), and Trifluoroacetyl (TFA).

Protecting/blocking groups are well known to those of skill as aremethods of coupling such groups to the appropriate residue(s) comprisingthe peptides of this invention (see, e.g., Greene et al., (1991)Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc.Somerset, N.J.). In one preferred embodiment, for example, acetylationis accomplished during the synthesis when the peptide is on the resinusing acetic anhydride. Amide protection can be achieved by theselection of a proper resin for the synthesis. During the synthesis ofthe peptides described herein in the examples, rink amide resin wasused. After the completion of the synthesis, the semi-permanentprotecting groups on acidic bifunctional amino acids such as Asp and Gluand basic amino acid Lys, hydroxyl of Tyr are all simultaneouslyremoved. The peptides released from such a resin using acidic treatmentcomes out with the n-terminal protected as acetyl and the carboxylprotected as NH₂ and with the simultaneous removal of all of the otherprotecting groups.

V. Enhancing Peptide Uptake.

It was also a surprising discovery of this invention that when an all Lamino acid peptide (e.g. otherwise having the sequence of the peptidesof this invention) is administered in conjunction with the D-form (i.e.a peptide of this invention) the uptake of the D-form peptide isincreased. Thus, in certain embodiments, this invention contemplates theuse of combinations of D-form and L-form peptides in the methods of thisinvention. The D-form peptide and the L-form peptide can have differentamino acid sequences, however, in preferred embodiments, they both haveamino acid sequences of peptides described herein, and in still morepreferred embodiments, they have the same amino acid sequence.

It was also a discovery of this invention that concatamers of theamphipathic helix peptides of this invention are also effective inmitigating one or more symptoms of atherosclerosis. The monomerscomprising the concatamers can be coupled directly together or joined bya linker. In certain embodiments, the linker is an amino acid linker(e.g. a proline), or a peptide linker (e.g. Gly₄Ser₃, SEQ ID NO:30). Incertain embodiments, the concatamer is a 2 mer, more preferably a 3 mer,still more preferably a 4 mer, and most preferably 5 mer, 8 mer or 10mer. As indicated above, the concatamer can comprise a G* relatedamphipathic helix as described herein combined with an apo A-I variantas described in PCT publication WO 02/15923.

VI. Pharmaceutical Formulations.

In order to carry out the methods of the invention, one or more peptidesor peptide mimetics of this invention are administered, e.g. to anindividual diagnosed as having one or more symptoms of atherosclerosis,or as being at risk for atherosclerosis. The peptides or peptidemimetics can be administered in the “native” form or, if desired, in theform of salts, esters, amides, prodrugs, derivatives, and the like,provided the salt, ester, amide, prodrug or derivative is suitablepharmacologically, i.e., effective in the present method. Salts, esters,amides, prodrugs and other derivatives of the active agents may beprepared using standard procedures known to those skilled in the art ofsynthetic organic chemistry and described, for example, by March (1992)Advanced Organic Chemistry; Reactions, Mechanisms and Structure, 4th Ed.N.Y. Wiley-Interscience.

For example, acid addition salts are prepared from the free base usingconventional methodology, that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or may be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include both organic acids, e.g., aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the peptides or mimetics areprepared in a similar manner using a pharmaceutically acceptable basesuch as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine, or the like. Particularly preferredbasic salts include alkali metal salts, e.g., the sodium salt, andcopper salts.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups which may be present within the molecularstructure of the drug. The esters are typically acyl-substitutedderivatives of free alcohol groups, i.e., moieties that are derived fromcarboxylic acids of the formula RCOOH where R is alky, and preferably islower alkyl. Esters can be reconverted to the free acids, if desired, byusing conventional hydrogenolysis or hydrolysis procedures.

Amides and prodrugs may also be prepared using techniques known to thoseskilled in the art or described in the pertinent literature. Forexample, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Prodrugs are typicallyprepared by covalent attachment of a moiety that results in a compoundthat is therapeutically inactive until modified by an individual'smetabolic system.

The peptides or mimetics identified herein are useful for parenteral,topical, oral, nasal (or otherwise inhaled), rectal, or localadministration, such as by aerosol or transdermally, for prophylacticand/or therapeutic treatment of atherosclerosis and/or symptoms thereof.The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. Suitable unitdosage forms, include, but are not limited to powders, tablets, pills,capsules, lozenges, suppositories, patches, nasal sprays, injectibles,implantable sustained-release formulations, lipid complexes, etc.

The peptides and/or peptide mimetics of this invention are typicallycombined with a pharmaceutically acceptable carrier (excipient) to forma pharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

The excipients are preferably sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well-knownsterilization techniques.

In therapeutic applications, the compositions of this invention areadministered to a patient suffering from one or more symptoms ofatherosclerosis or at risk for atherosclerosis in an amount sufficientto cure or at least partially prevent or arrest the disease and/or itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of the active agents of the formulationsof this invention to effectively treat (ameliorate one or more symptoms)the patient.

The concentration of peptide or mimetic can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs. Concentrations, however, willtypically be selected to provide dosages ranging from about 0.1 or 1mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosagesrange from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably fromabout 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferredembodiments, dosages range from about 10 mg/kg/day to about 50mg/kg/day. In certain embodiments, dosages range from about 20 mg toabout 50 mg given orally twice daily. It will be appreciated that suchdosages may be varied to optimize a therapeutic regimen in a particularsubject or group of subjects.

In certain preferred embodiments, the peptides or peptide mimetics ofthis invention are administered orally (e.g. via a tablet) or as aninjectable in accordance with standard methods well known to those ofskill in the art. In other preferred embodiments, the peptides, may alsobe delivered through the skin using conventional transdermal drugdelivery systems, i.e., transdermal “patches” wherein the activeagent(s) are typically contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the drug composition is typically contained in a layer, or“reservoir,” underlying an upper backing layer. It will be appreciatedthat the term “reservoir” in this context refers to a quantity of“active ingredient(s)” that is ultimately available for delivery to thesurface of the skin. Thus, for example, the “reservoir” may include theactive ingredient(s) in an adhesive on a backing layer of the patch, orin any of a variety of different matrix formulations known to those ofskill in the art. The patch may contain a single reservoir, or it maycontain multiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

Other preferred formulations for topical drug delivery include, but arenot limited to, ointments and creams. Ointments are semisolidpreparations which are typically based on petrolatum or other petroleumderivatives. Creams containing the selected active agent, are typicallyviscous liquid or semisolid emulsions, often either oil-in-water orwater-in-oil. Cream bases are typically water-washable, and contain anoil phase, an emulsifier and an aqueous phase. The oil phase, alsosometimes called the “internal” phase, is generally comprised ofpetrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil phasein volume, and generally contains a humectant. The emulsifier in a creamformulation is generally a nonionic, anionic, cationic or amphotericsurfactant. The specific ointment or cream base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery. As with other carriers or vehicles, an ointmentbase should be inert, stable, nonirritating and nonsensitizing.

Unlike typical peptide formulations, the peptides of this inventioncomprising D-form amino acids can be administered, even orally, withoutprotection against proteolysis by stomach acid, etc. Nevertheless, incertain embodiments, peptide delivery can be enhanced by the use ofprotective excipients. This is typically accomplished either bycomplexing the polypeptide with a composition to render it resistant toacidic and enzymatic hydrolysis or by packaging the polypeptide in anappropriately resistant carrier such as a liposome. Means of protectingpolypeptides for oral delivery are well known in the art (see, e.g.,U.S. Pat. No. 5,391,377 describing lipid compositions for oral deliveryof therapeutic agents).

Elevated serum half-life can be maintained by the use ofsustained-release protein “packaging” systems. Such sustained releasesystems are well known to those of skill in the art. In one preferredembodiment, the ProLease biodegradable microsphere delivery system forproteins and peptides (Tracy (1998) Biotechnol. Prog. 14: 108; Johnsonet al. (1996), Nature Med. 2: 795; Herbert et al. (1998), Pharmaceut.Res. 15, 357) a dry powder composed of biodegradable polymericmicrospheres containing the protein in a polymer matrix that can becompounded as a dry formulation with or without other agents.

The ProLease microsphere fabrication process was specifically designedto achieve a high protein encapsulation efficiency while maintainingprotein integrity. The process consists of (i) preparation offreeze-dried protein particles from bulk protein by spray freeze-dryingthe drug solution with stabilizing excipients, (ii) preparation of adrug-polymer suspension followed by sonication or homogenization toreduce the drug particle size, (iii) production of frozen drug-polymermicrospheres by atomization into liquid nitrogen, (iv) extraction of thepolymer solvent with ethanol, and (v) filtration and vacuum drying toproduce the final dry-powder product. The resulting powder contains thesolid form of the protein, which is homogeneously and rigidly dispersedwithin porous polymer particles. The polymer most commonly used in theprocess, poly(lactide-co-glycolide) (PLG), is both biocompatible andbiodegradable.

Encapsulation can be achieved at low temperatures (e.g., −40° C.).During encapsulation, the protein is maintained in the solid state inthe absence of water, thus minimizing water-induced conformationalmobility of the protein, preventing protein degradation reactions thatinclude water as a reactant, and avoiding organic-aqueous interfaceswhere proteins may undergo denaturation. A preferred process usessolvents in which most proteins are insoluble, thus yielding highencapsulation efficiencies (e.g., greater than 95%).

In another embodiment, one or more components of the solution can beprovided as a “concentrate”, e.g., in a storage container (e.g., in apremeasured volume) ready for dilution, or in a soluble capsule readyfor addition to a volume of water.

The foregoing formulations and administration methods are intended to beillustrative and not limiting. It will be appreciated that, using theteaching provided herein, other suitable formulations and modes ofadministration can be readily devised.

VII. Lipid-Based Formulations.

In certain embodiments, the peptides of this invention are administeredin conjunction with one or more lipids. The lipids can be formulated asan excipient to protect and/or enhance transport/uptake of the peptidesor they can be administered separately.

Without being bound by a particular theory, it was discovered of thisinvention that administration (e.g. oral administration) of certainphospholipids can significantly increase HDL/LDL ratios. In addition, itis believed that certain medium-length phospholipids are transported bya process different than that involved in general lipid transport. Thus,co-administration of certain medium-length phospholipids with thepeptides of this invention confer a number of advantages: They protectthe phospholipids from digestion or hydrolysis, they improve peptideuptake, and they improve HDL/LDL ratios.

The lipids can be formed into liposomes that encapsulate thepolypeptides of this invention and/or they can be complexed/admixed withthe polypeptides and/or they can be covalently coupled to thepolypeptides. Methods of making liposomes and encapsulating reagents arewell known to those of skill in the art (see, e.g., Martin andPapahadjopoulos (1982) J. Biol. Chem., 257: 286-288; Papahadjopoulos etal. (1991) Proc. Natl. Acad. Sci. USA, 88: 11460-11464; Huang et al.(1992) Cancer Res., 52: 6774-6781; Lasic et al. (1992) FEBS Lett., 312:255-258., and the like).

Preferred phospholipids for use in these methods have fatty acidsranging from about 4 carbons to about 24 carbons in the sn-1 and sn-2positions. In certain preferred embodiments, the fatty acids aresaturated. In other preferred embodiments, the fatty acids can beunsaturated. Various preferred fatty acids are illustrated in Table 3.

TABLE 3 Preferred fatty acids in the sn-1 and/or sn-2 position of thepreferred phospholipids for admission of D polypeptides. Carbon No.Common Name IUPAC Name  3:0 Propionoyl Trianoic  4:0 Butanoyl Tetranoic 5:0 Pentanoyl Pentanoic  6:0 Caproyl Hexanoic  7:0 Heptanoyl Heptanoic 8:0 Capryloyl Octanoic  9:0 Nonanoyl Nonanoic 10:0 Capryl Decanoic 11:0Undcanoyl Undecanoic 12:0 Lauroyl Dodecanoic 13:0 TridecanoylTridecanoic 14:0 Myristoyl Tetradecanoic 15:0 PentadecanoylPentadecanoic 16:0 Palmitoyl Hexadecanoic 17:0 HeptadecanoylHeptadecanoic 18:0 Stearoyl Octadecanoic 19:0 Nonadecanoyl Nonadecanoic20:0 Arachidoyl Eicosanoic 21:0 Heniecosanoyl Heniecosanoic 22:0Behenoyl Docosanoic 23:0 Truisanoyl Trocosanoic 24:0 LignoceroylTetracosanoic 14:1 Myristoleoyl (9-cis) 14:1 Myristelaidoyl (9- trans)16:1 Palmitoleoyl (9-cis) 16:1 Palmitelaidoyl (9- trans)The fatty acids in these positions can be the same or different.Particularly preferred phospholipids have phosphorylcholine at the sn-3position.VIII. Additional Pharmacologically Active Agents.

Additional pharmacologically active agents may be delivered along withthe primary active agents, e.g., the peptides of this invention. In oneembodiment, such agents include, but are not limited to agents thatreduce the risk of atherosclerotic events and/or complications thereof.Such agents include, but are not limited to beta blockers, beta blockersand thiazide diuretic combinations, statins, aspirin, ace inhibitors,ace receptor inhibitors (ARBs), and the like.

Suitable beta blockers include, but are not limited to cardioselective(selective beta 1 blockers), e.g., acebutolol (Sectral™), atenolol(Tenormin™), betaxolol (Kerlone™), bisoprolol (Zebeta™), metoprolol(Lopressor™), and the like. Suitable non-selective blockers (block beta1 and beta 2 equally) include, but are not limited to carteolol(Cartrol™), nadolol (Corgard™), penbutolol (Levatol™), pindolol(Visken™), propranolol (Inderal™), timolol (Blockadren™), labetalol(Normodyne™, Trandate™), and the like.

Suitable beta blocker thiazide diuretic combinations include, but arenot limited to Lopressor HCT, ZIAC, Tenoretic, Corzide, Timolide,Inderal LA 40/25, Inderide, Normozide, and the like.

Suitable statins include, but are not limited to pravastatin(Pravachol/Bristol-Myers Squibb), simvastatin (Zocor/Merck), lovastatin(Mevacor/Merck), and the like.

Suitable ace inhibitors include, but are not limited to captopril (e.g.Capoten™ by Squibb), benazepril (e.g., Lotensin™ by Novartis), enalapril(e.g., Vasotec™ by Merck), fosinopril (e.g., Monopril™ byBristol-Myers), lisinopril (e.g. Prinivil™ by Merck or Zestril™ byAstra-Zeneca), quinapril (e.g. Accupril™ by Parke-Davis), ramipril(e.g., Altace™ by Hoechst Marion Roussel, King Pharmaceuticals),imidapril, perindopril erbumine (e.g., Aceon™ by Rhone-Polenc Rorer),trandolapril (e.g., Mavik™ by Knoll Pharmaceutical), and the like.Suitable ARBS (Ace Receptor Blockers) include but are not limited tolosartan (e.g. Cozaar™ by Merck), irbesartan (e.g., Avapro™ by Sanofi),candesartan (e.g., Atacand™ by Astra Merck), valsartan (e.g., Diovan™ byNovartis), and the like.

IX. Kits for the Amelioration of One or More Symptoms ofAtherosclerosis.

In another embodiment this invention provides kits for amelioration ofone or more symptoms of atherosclerosis or for the prophylactictreatment of a subject (human or animal) at risk for atherosclerosis.The kits preferably comprise a container containing one or more of thepeptides or peptide mimetics of this invention. The peptide or peptidemimetic can be provided in a unit dosage formulation (e.g. suppository,tablet, caplet, patch, etc.) and/or may be optionally combined with oneor more pharmaceutically acceptable excipients.

The kit can, optionally, further comprise one or more other agents usedin the treatment of heart disease and/or atherosclerosis. Such agentsinclude, but are not limited to, beta blockers, vasodilators, aspirin,statins, ace inhibitors or ace receptor inhibitors (ARBs) and the like,e.g. as described above.

In addition, the kits optionally include labeling and/or instructionalmaterials providing directions (i.e., protocols) for the practice of themethods or use of the “therapeutics” or “prophylactics” of thisinvention. Preferred instructional materials describe the use of one ormore polypeptides of this invention to mitigate one or more symptoms ofatherosclerosis and/or to prevent the onset or increase of one or moreof such symptoms in an individual at risk for atherosclerosis and/or tomitigate one or more symptoms of a pathology characterized by aninflammatory response. The instructional materials may also, optionally,teach preferred dosages/therapeutic regiment, counter indications andthe like.

While the instructional materials typically comprise written or printedmaterials they are not limited to such. Any medium capable of storingsuch instructions and communicating them to an end user is contemplatedby this invention. Such media include, but are not limited to electronicstorage media (e.g., magnetic discs, tapes, cartridges, chips), opticalmedia (e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Use of ApoJ-Related Peptides to Mediate Symptoms ofAtherosclerosis

Prevention of LDL-Induced Monocyte Chemotactic Activity

FIG. 1 illustrates a comparison of the effect of D-4F (Circulation2002;105:290-292) with the effect of an apoJ peptide made from D aminoacids (D-J336, Ac-LLEQLNEQFNWVSRLANLTEGE-NH₂, SEQ ID NO:1) on theprevention of LDL-induced monocyte chemotactic activity in vitro in aco-incubation. Human aortic endothelial cells were incubated with mediumalone (no addition), with control human LDL (200 μg protein/ml) orcontrol human LDL+control human HDL (350 μg HDL protein/ml). D-J336 orD-4F was added to other wells in a concentration range as indicated pluscontrol human LDL (200 μg protein/ml). Following overnight incubation,the supernatants were assayed for monocyte chemotactic activity. Asshown in FIG. 1, the in vitro concentration of the apoJ variant peptidethat prevents LDL-induced monocyte chemotactic activity by human arterywall cells is 10 to 25 times less than the concentration required forthe D-4F peptide.

Prevention of LDL-Induced Monocyte Chemotactic Activity by Pre-Treatmentof Artery Wall Cells with D-J336

FIG. 2 illustrates a comparison of the effect of D-4F with the effect ofD-J336 on the prevention of LDL induced monocyte chemotactic activity ina pre-incubation. Human aortic endothelial cells were pre-incubated withD-J336 or D-4F at 4, 2, and 1 μg/ml for DJ336 or 100, 50, 25, and 12.5μg/ml for D-4F for 6 hrs. The cultures were then washed and wereincubated with medium alone (no addition), or with control human LDL(200 μg protein/ml), or with control human LDL+control human HDL (350 μgHDL protein/ml) as assay controls. The wells that were pre-treated withpeptides received the control human LDL at 200 μg protein/ml. Followingovernight incubation, the supernatants were assayed for monocytechemotactic activity.

As illustrated in FIG. 2, the ApoJ variant peptide was 10-25 times morepotent in preventing LDL oxidation by artery wall cells in vitro.

The Effect of Apo J Peptide Mimetics on HDL Protective Capacity in LDLReceptor Null Mice.

D-4F designated as F, or the apoJ peptide made from D amino acids(D-J336, designated as J) was added to the drinking water of LDLreceptor null mice (4 per group) at 0.25 or 0.5 mg per ml of drinkingwater. After 24- or 48-hrs blood was collected from the mice and theirHDL was isolated and tested for its ability to protect againstLDL-induced monocyte chemotactic activity. Assay controls includedculture wells that received no lipoproteins (no addition), or controlhuman LDL alone (designated as LDL, 200 μg cholesterol/ml), or controlLDL+control human HDL (designated as +HDL, 350 μg HDL cholesterol). Fortesting the mouse HDL, the control LDL was added together with mouse HDL(+F HDL or +J HDL) to artery wall cell cultures. The mouse HDL was addedat 100 μg cholesterol/ml respectively. After treatment with either D-4For D-J336 the mouse HDL at 100 μg/ml was as active as 350 μg/ml ofcontrol human HDL in preventing the control LDL from inducing the arterywall cells to produce monocyte chemotactic activity. The reason for thediscrepancy between the relative doses required for the D-J336 peptiderelative to D-4F in vitro and in vivo may be related to the solubilityof the peptides in water and we believe that when measures are taken toachieve equal solubility the D-J peptides will be much more active invivo as they are in vitro.

Protection Against LDL-Induced Monocyte Chemotactic Activity by HDL fromapo E Null Mice given Oral Peptides.

FIG. 4 illustrates the effect of oral apoA-1 peptide mimetic and Apojpeptide on HDL protective capacity. ApoE null mice (4 per group) wereprovided with D-4F (designated as F) at 50, 30, 20, 10, 5 μg per ml ofdrinking water or apoJ peptide (designated as J) at 50, 30 or 20 μg perml of drinking water. After 24 hrs blood was collected, plasmafractionated by FPLC and fractions containing LDL (designated as mlDLfor murine LDL) and fractions containing HDL (designated as mHDL) wereseparately pooled and HDL protective capacity against LDL oxidation asdetermined by LDL-induced monocyte chemotactic activity was determined.For the assay controls the culture wells received no lipoproteins (noadditions), mlDL alone (at 200 μg cholesterol/ml), or mlDL+standardnormal human HDL (designated as Cont. h HDL, at 350 μg HDLcholesterol/ml).

For testing the murine HDL, mlDL together with murine HDL (+F mHDL or +JmHDL) were added to artery wall cell cultures. The HDL from the micethat did not receive any peptide in their drinking water is designatedas no peptide mHDL. The murine HDL was used at 100 μg cholesterol/ml.After receiving D-4F or D-J336 the murine HDL at 100 μg/ml was as activeas 350 μg/ml of normal human HDL. As shown in FIG. 4, when added to thedrinking water the D-J peptide was as potent as D-4F in enhancing HDLprotective capacity in apo E null mice.

Ability of LDL Obtained from apoE Null Mice Given Oral Peptides toInduce Monocyte Chemotactic Activity.

FIG. 5 illustrates the effect of oral apo A-1 peptide mimetic and apoJpeptide on LDL susceptibility to oxidation. ApoE null mice (4 per group)were provided, in their drinking water, with D-4F (designated as F) at50, 30, 20, 10, 5 μg per ml of drinking water or the apoJ peptide(D-J336 made from D amino acids and designated as J) at 50, 30 or 20 μgper ml of drinking water. After 24 hrs blood was collected from the miceshown in FIG. 4, plasma fractionated by FPLC and fractions containingLDL (designated as mlDL for murine LDL) were pooled and LDLsusceptibility to oxidation as determined by induction of monocytechemotactic activity was determined. For the assay controls the culturewells received no lipoproteins (no additions), mlDL alone (at 200 μgcholesterol/ml), or mlDL+standard normal human HDL (designated as Cont.h HDL, 350 μg HDL cholesterol).

Murine LDL, mlDL, from mice that received the D-4F (F mlDL) or thosethat received the apoJ peptide (J mlDL) were added to artery wall cellcultures. LDL from mice that did not receive any peptide in theirdrinking water is designated as No peptide LDL.

As shown in FIG. 5, when added to the drinking water, D-J336 wasslightly more potent than D-4F in rendering the LDL from apo E null miceresistant to oxidation by human artery wall cells as determined by theinduction of monocyte chemotactic activity.

Protection Against Phospholipid Oxidation and Induction of MonocyteChemotactic Activity by HDL Obtained from apo E Null Mice Given OralPeptides.

FIG. 6 illustrates the effect of oral apoA-1 peptide mimetic and apojpeptide on HDL protective capacity. ApoE null mice (4 per group) wereprovided with D-4F (designated as F) at 50, 30, 20, 10, 5 μg per ml ofdrinking water or apoj peptide (D-J336 made from D amino acids anddesignated as J) at 50, 30 or 20 μg per ml of drinking water. After 24hrs blood was collected, plasma fractionated by FPLC and fractionscontaining HDL (designated as mHDL) were pooled and HDL protectivecapacity against PAPC oxidation as determined by the induction ofmonocyte chemotactic activity was determined. For the assay controls theculture wells received no lipoproteins (no additions), the phospholipidPAPC at 20 μg/ml+HPODE, at 1.0 μg/ml, or PAPC+HPODE plus standard normalhuman HDL (at 350 μg HDL cholesterol/ml and designated as +Cont. h HDL).

For testing the murine HDL, PAPC+HPODE together with murine HDL (+F mHDLor +J mHDL) were added to artery wall cell cultures. The HDL from micethat did not receive any peptide in their drinking water is designatedas “no peptide mHDL”. The murine HDL was used at 100 μg cholesterol/ml.

The data show in FIG. 6 indicate that, when added to the drinking water,D-J336 was as potent as D-4F in causing HDL to inhibit the oxidation ofa phospholipid PAPC by the oxidant HPODE in a human artery wallco-culture as measured by the generation of monocyte chemotacticactivity

Effect of Oral apoA-1 Peptide Mimetic and apoJ Peptide on PlasmaParaoxonase Activity in Mice.

FIG. 7 shows the effect of oral apoA-1 peptide mimetic and apoj peptideon plasma paraoxonase activity in mice. ApoE null mice (4 per group)were provided with D-4F designated as F at 50, 10, 5 or 0 μg per ml ofdrinking water or apoJ peptide (D-J336 made from D amino acids anddesignated as J) at 50, 10 or 5 μg per ml of drinking water. After 24hrs blood was collected and plasma was assayed for PON1 activity. Thesedata demonstrate that, when added to the drinking water, D-J336 was atleast as potent as D-4F in increasing the paraoxonase activity of apo Enull mice.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1. An isolated polypeptide that ameliorates a symptom of atherosclerosisor other pathology associated with an inflammatory response, saidpolypeptide comprising an amphipathic helical peptide having chargedresidues on the polar face of the peptide and possessing a widenon-polar face, wherein said peptide comprises an amino acid sequenceselected from the group consisting of LLEQLNEQFNWVSRLANL (SEQ ID NO:2),and LVGRQLEEFL, (SEQ ID NO:9), and wherein said polypeptide is 40 orfewer amino acids in length and at least 10 amino acids in length. 2.The polypeptide of claim 1, wherein said peptide comprises a G*amphipathic helix.
 3. The polypeptide of claim 2, wherein said peptideshows greater than about 50% sequence identity with apo J.
 4. Thepolypeptide of claim 1, wherein said peptide protects a phospholipidagainst oxidation by an oxidizing agent.
 5. The polypeptide of claim 1,wherein said peptide is a concatamer of two or more of said amino acidsequences.
 6. The polypeptide of claim 1, wherein said peptide furthercomprises a protecting group.
 7. The polypeptide of claim 1, whereinsaid peptide further comprises a protecting group coupled to the aminoor carboxyl terminus.
 8. The polypeptide of claim 6, wherein saidprotecting group is a protecting group selected from the groupconsisting of an acetyl, amide, 3 to 20 carbon alkyl groups, Fmoc,9-fluoreneacetyl group, 1-fluorenecarboxylic group,9-fluorenecarboxylic, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mint), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl(Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),1-(4,4dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO),t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), a propyl group, a butyl group, a pentylgroup, a hexyl group, and Trifluoroacetyl (TFA).
 9. The polypeptide ofclaim 6, wherein said peptide comprises a protecting group coupled tothe amino terminus and said amino terminal protecting group is aprotecting group selected from the group consisting of a benzoyl group,an acetyl, a propionyl, a carbobenzoxy, a propyl, a butyl, a pentyl, ahexyl, and a 3 to 20 carbon alkyl.
 10. The polypeptide of claim 6,wherein said peptide comprises a protecting group coupled to thecarboxyl terminus and said carboxyl terminal protecting group is anamide.
 11. The polypeptide of claim 6, wherein said peptide furthercomprises: a first protecting group coupled to the amino terminuswherein said protecting group is a protecting group selected from thegroup consisting of a benzoyl group, an acetyl, a propionyl, acarbobenzoxy, a propyl, a butyl, a pentyl, a hexyl, and a 3 to 20 carbonalkyl; and a second protecting group coupled to the carboxyl terminusand said carboxyl terminal protecting group is an amide.
 12. Thepolypeptide of claim 1, wherein said peptide comprises a firstprotecting group coupled to the amino terminus and a second protectinggroup coupled to the carboxyl terminus.
 13. The polypeptide of claim 1,wherein said polypeptide comprises an Ac group on the amino terminus andan —NH₂ on the carboxyl terminus.
 14. The polypeptide of claim 1,wherein said peptide comprises a “D” amino acid.
 15. The polypeptide ofclaim 1, wherein said peptide comprises a plurality of “D” amino acids.16. The polypeptide of claim 1, wherein all enantiomeric amino acidscomprising said peptide are “D” amino acids.
 17. The polypeptide ofclaim 1, wherein said polypeptide is mixed with a pharmacologicallyacceptable excipient.
 18. The polypeptide of claim 1, wherein saidpolypeptide is mixed with a pharmacologically acceptable excipientsuitable for oral administration to a mammal.
 19. An isolatedpolypeptide that ameliorates a symptom of atherosclerosis or otherpathology associated with an inflammatory response, said polypeptidecomprising an amphipathic helical peptide having charged residues on thepolar face of the peptide and possessing a wide non-polar face, whereinsaid polypeptide is coupled to a phospholipid.
 20. The polypeptide ofclaim 19, wherein said polypeptide is covalently coupled to aphospholipid.
 21. The polypeptide of claim 19, wherein said polypeptideis covalently coupled to a phospholipid comprising lysophosphatidylcholine.
 22. The polypeptide of claim 19, wherein said polypeptide iscovalently coupled to a phospholipid comprising a fatty acid selectedfrom the group consisting of propionoyl, butanoyl, pentanoyl, caproyl,heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl,tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, heptadecanoyl,stearoyl, nonadecanoyl, arachidoyl, heniecosanoyl, behenoyl,trucisanoyl, lignoceroyl, myristoleoyl (9-cis), myristelaidoyl(9-trans), palmitoleoyl (9-cis), and palmitelaidoyl (9-trans).
 23. Thepolypeptide of claim 22, wherein said polypeptide is covalently coupledto the sn-1 or sn-2 position of said phospholipid.
 24. A compositionsuitable for oral administration that ameliorates a symptom ofatherosclerosis, wherein said composition comprises a peptide comprisingan amphipathic helix having charged residues on the polar face of thepeptide and possessing a wide non-polar face, wherein said peptidecomprises a D amino acid and said peptide is blocked at the aminoterminus and the carboxyl terminus.
 25. The composition of claim 24,wherein said peptide is at least 10 amino acids in length.
 26. Thecomposition of claim 25, wherein said peptide is about 40 or fewerpeptides in length.
 27. The composition of claim 25, wherein saidpeptide comprises a G* amphipathic helix.
 28. The composition of claim27, wherein said peptide shows greater than about 50% sequence identitywith apo J.
 29. The composition of claim 25, wherein said peptideprotects a phospholipid against oxidation by an oxidizing agent.
 30. Thecomposition of claim 25, wherein said peptide comprises an amino acidsequence selected from the group consisting of LLEQLNEQFNWVSRLANL (SEQID NO:2), and LVGRQLEEFL (SEQ ID NO:9).
 31. The composition of claim 25,wherein said peptide is blocked at the amino terminus with a firstprotecting group and said peptide is blocked at the carboxyl terminuswith a second protecting group and said first protecting group and saidsecond protecting group are independently selected from the groupconsisting of an acetyl, amide, 3 to 20 carbon alkyl groups, Fmoc,9-fluoreneacetyl group, 1-fluorenecarboxylic group,9-fluorenecarboxylic, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl (Mtr),Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl(Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl(MeBzl), 4-methoxyberizyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl),Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCI-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), a propyl group, a butyl group, a pentylgroup, a hexyl group, and Trifluoroacetyl (TFA).
 32. The composition ofclaim 25, wherein said first protecting group is an acetyl.
 33. Thecomposition of claim 25, wherein said second protecting group is anamide.
 34. The composition of claim 25, wherein more than half of theenantiomeric amino acids comprising said peptide are D amino acids. 35.The composition of claim 25, wherein all enantiomeric amino acidscomprising said peptide are D amino acids.
 36. The composition of claim25, wherein said composition further comprises a pharmaceuticallyacceptable excipient.
 37. The composition of claim 36, wherein saidexcipient is an excipient suitable for oral administration.
 38. Thecomposition of claim 36, wherein said excipient is an excipient suitablefor injection.
 39. A pharmaceutical composition, said compositioncomprising a polypeptide of any of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, or 18 in a pharmaceutically acceptableexcipient.
 40. The composition of claim 39, wherein said composition isin the form of a unit dosage formulation.
 41. The composition of claim25, wherein said peptide is coupled to a phospholipid.
 42. Thecomposition of claim 41, wherein said peptide is covalently coupled to aphospholipid.
 43. The composition of claim 41, wherein said peptide iscovalently coupled to a phospholipid comprising lysophosphatidylcholine.
 44. The composition of claim 41, wherein said peptide iscovalently coupled to a phospholipid comprising a fatty acid selectedfrom the group consisting of propionoyl, butanoyl, pentanoyl, caproyl,heptanoyl, capryloyl, nonanoyl, capryl, undecanoyl, lauroyl,tridecanoyl, myristoyl, pentadecanoyl, palmitoyl, heptadecanoyl,stearoyl, nonadecanoyl, arachidoyl, heniecosanoyl, behenoyl,trucisanoyl, lignoceroyl, myristoleoyl (9-cis), myristelaidoyl(9-trans), palmitoleoyl (9-cis), and palmitelaidoyl (9-trans).
 45. Thepolypeptide of claim 19, wherein said peptide is at least 10 amino acidsin length.
 46. The polypeptide of claim 19, wherein said peptide isabout 40 or fewer amino acids in length.
 47. The polypeptide of claim19, wherein said peptide comprises a G* amphipathic helix.
 48. Thepolypeptide of claim 47, wherein said peptide shows greater than about50% sequence identity with apo J.
 49. The polypeptide of claim 19,wherein said peptide comprises an amino acid sequence selected from thegroup consisting of LLEQLNEQFNWVSRLANL (SEQ ID NO:2), and LVGRQLEEFL(SEQ ID NO:9).
 50. The polypeptide of claim 49, wherein said peptide isa concatamer of two or more of said amino acid sequences.
 51. Thepolypeptide of claim 19, wherein said peptide further comprises aprotecting group coupled to the amino or carboxyl terminus.
 52. Thepolypeptide of claim 51, wherein said protecting group is a protectinggroup selected from the group consisting of an acetyl, amide, 3 to 20carbon alkyl groups, Fmoc, 9-fluoreneacetyl group, 1-fluorenecarboxylicgroup, 9-fluorenecarboxylic, 9-fluorenone-1-carboxylic group,benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4-methyltrityl (Mtt),4-methoxytrityl (Mint), 4-methoxy-2,3,6-trimethyl-benzenesulphonyl(Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh),Tosyl (Tos), 2,2,5,7,8-pentamethyl chroman-6-sulphonyl (Pmc),4-methylbenzyl (MeBzl), 4-methoxybenzyl (MeOBzl), Benzyloxy (BzlO),Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2-pyridinesulphenyl (Npys),1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), 2,6-dichlorobenzyl(2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-Cl-Z),2-bromobenzyloxycarbonyl (2-Br-Z), Benzyloxymethyl (Bom),t-butoxycarbonyl (Boc), cyclohexyloxy (cHxO), t-butoxymethyl (Bum),t-butoxy (tBuO), t-Butyl (tBu), a propyl group, a butyl group, a pentylgroup, a hexyl group, and Trifluoroacetyl (TFA).
 53. The polypeptide ofclaim 51, wherein said peptide comprises a protecting group coupled tothe amino terminus and said amino terminal protecting group is aprotecting group selected from the group consisting of a benzoyl group,an acetyl, a propionyl, a carbobenzoxy, a propyl, a butyl, a pentyl, ahexyl, and a 3 to 20 carbon alkyl.
 54. The polypeptide of claim 51,wherein said peptide comprises a protecting group coupled to thecarboxyl terminus and said carboxyl terminal protecting group is anamide.
 55. The polypeptide of claim 51, wherein said peptide furthercomprises: a first protecting group coupled to the amino terminuswherein said protecting group is a protecting group selected from thegroup consisting of a benzoyl group, an acetyl, a propionyl, acarbobenzoxy, a propyl, a butyl, a pentyl, a hexyl, and a 3 to 20 carbonalkyl; and a second protecting group coupled to the carboxyl terminusand said carboxyl terminal protecting group is an amide.
 56. Thepolypeptide of claim 51, wherein said peptide comprises a firstprotecting group coupled to the amino terminus and a second protectinggroup coupled to the carboxyl terminus.
 57. The polypeptide of claim 51,wherein said peptide comprises an Ac group on the amino terminus. 58.The polypeptide of claim 51, wherein said peptide comprises an —NH₂ onthe carboxyl terminus.
 59. The polypeptide of claim 51, wherein saidpeptide comprises an Ac group on the amino terminus and an —NH₂ on thecarboxyl terminus.
 60. The polypeptide of claim 51, wherein said peptidecomprises a “D” amino acid.
 61. The polypeptide of claim 51, whereinsaid peptide comprises a plurality of “D” amino acids.
 62. Thepolypeptide of claim 51, wherein all enantiomeric amino acids comprisingsaid peptide are “D” amino acids.
 63. A method of ameliorating a symptomof atherosclerosis in a mammal, said method comprising administering tosaid mammal a polypeptide according to any of claims 1, 2, 3, 4, 5-12,13, 16, 19-23, and 45
 62. 64. The method of claim 63, wherein saidadministering comprises orally administering said polypeptide.
 65. Themethod of claim 63, wherein said mammal is a mammal diagnosed as havingone or more symptoms of atherosclerosis.
 66. The method of claim 63,wherein said mammal is a mammal diagnosed as at risk foratherosclerosis.
 67. The method of claim 63, wherein said mammal is ahuman.
 68. The method of claim 63, wherein said mammal is non-humanmammal.
 69. A method of ameliorating a symptom of a pathologycharacterized by an inflammatory response in a mammal, said methodcomprising administering to said mammal a polypeptide according to anyof claims 1, 2, 3, 4, 5-12, 13-16, 19-23, and 45-62.
 70. The method ofclaim 69, wherein said mammal is a mammal diagnosed as having one ormore symptoms of an inflammatory response.
 71. The method of claim 69,wherein said mammal is a mammal diagnosed as at risk for a pathologyassociated with an inflammatory response.
 72. The method of claim 69,wherein said mammal is a human.
 73. The method of claim 69, wherein saidmammal is a non-human mammal.
 74. A kit for ameliorating a symptom ofatherosclerosis, said kit comprising a container containing apolypeptide according to any of claims 2, 3, 4, 5-12, 13-18, 19-23, and45-62.
 75. The kit of claim 74, wherein said polypeptide is combinedwith a pharmaceutically acceptable excipient in a unit dosageformulation.
 76. The kit of claim 75, wherein said unit dosageformulation is for oral administration.
 77. The kit of claim 74, furthercomprising instructional materials teaching the use of said polypeptideor said composition for ameliorating one or more symptoms ofatherosclerosis or of a pathology characterized by an inflammatoryresponse.
 78. A method of mitigating or preventing a coronarycomplication associated with an acute phase response to an inflammationin a mammal, wherein said coronary complication is a symptom ofatherosclerosis, said method comprising administering to a mammal havingsaid acute phase response or at risk for said acute phase response, apolypeptide according to any of claims 1, 2, 3, 4, 5-12, 13-16, 19-23,and 45-62.
 79. The method of claim 78, said administration is by a routeselected from the group consisting of oral administration, nasaladministration, rectal administration, intraperitoneal injection,intravascular injection, subcutaneous injection, transcutaneousadministration, and intramuscular injection.
 80. The method of claim 78,wherein said polypeptide is administered in combination with an allL-form of the same polypeptide.
 81. The method of claim 78, wherein saidpolypeptide is provided as a unit formulation in a pharmaceuticallyacceptable excipient.
 82. The method of claim 78, wherein said acutephase response is an inflammatory response associated with a recurrentinflammatory disease.
 83. The method of claim 79, wherein said acutephase response is associated with a disease selected from the groupconsisting of leprosy, tuberculosis, systemic lupus erythematosus,polymyalgia rheumatica, polyarteritis nodosa, scleroderma, idiopathicpulmonary fibrosis, chronic obstructive pulmonary disease, coronarycalcification, calcific aortic stenosis, osteoporosis, and rheumatoidarthritis.
 84. The method of claim 78, wherein said acute phase responseis an inflammatory response associated with a condition selected fromthe group consisting of a bacterial infection, a viral infection, afungal infection, an organ transplant, a wound, an implanted prosthesis,parasitic infection, sepsis, endotoxic shock syndrome, and biofilmformation.
 85. A method of mitigating or preventing a coronarycomplication associated with an acute phase response to an inflammationin a mammal, wherein said coronary complication is a symptom ofatherosclerosis, said method comprising: assaying said mammal for anacute phase protein (APP) level indicative of an acute phase response ora significant risk of an acute phase response; and administering to amammal showing an acute phase protein (APP) level indicative of an acutephase response a polypeptide according to any of claims 1, 2, 3, 4,5-12, 13-16, 19-23, and 45-62.
 86. The method of claim 85, wherein saidacute phase protein (APP) is a postive APR selected from the groupconsisting of serum amyloid A, c-reactive protein, serum amyloid Pcomponent, C2 complement protein, C3 complement protein, C4 complementprotein, C5 complement protein, C9 complement protein, B complementprotein, C1 inhibitor, C4 binding protein, fibrinogen, von Willebrandfactor, α1-antitrypsin, α1-antichymotrypsin, α2 antiplasmin, heparincofactor II, plasminogen activator inhibitor I, haptoglobin, haemopexin,ceruloplasmin , manganese superoxide dismutase, α1-acid glycoprotein,haeme oxygenase, mannose binding protein, leukocyte protein I,lipoprotein (a), and lipopolysaccharide binding protein.
 87. The methodof claim 85, wherein said acute phase protein (APP) is a negative APRselected from the group consisting of albumin, prealbumin, transferin,apoAI, apoAII, α2-HS glycoprotein, inter-α-trypsin inhibitor, andhistidine-rich glycoprotein.
 88. The method of claim 63, wherein saidpolypeptide is in a pharmaceutically acceptable excipient.
 89. Themethod of claim 63, wherein said polypeptide is in a pharmaceuticallyacceptable excipient suitable for oral administration to a mammal. 90.The method of claim 63, wherein said polypeptide is in apharmaceutically acceptable excipient in the form of a unit dosageformulation.
 91. The method of claim 69, wherein said polypeptide is ina pharmaceutically acceptable excipient.
 92. The method of claim 69,wherein said polypeptide is in a pharmaceutically acceptable excipientsuitable for oral administration to a mammal.
 93. The method of claim69, wherein said polypeptide is in a pharmaceutically acceptableexcipient in the form of a unit dosage formulation.
 94. The kit of claim74, wherein said polypeptide is in a pharmaceutically acceptableexcipient.
 95. The kit of claim 74, wherein said polypeptide is in apharmaceutically acceptable excipient suitable for oral administrationto a mammal.
 96. The kit method of claim 74, wherein said polypeptide isin a pharmaceutically acceptable excipient in the form of a unit dosageformulation.
 97. The method of claim 78, wherein said polypeptide is ina pharmaceutically acceptable excipient.
 98. The method of claim 78,wherein said polypeptide is in a pharmaceutically acceptable excipientsuitable for oral administration to a mammal.
 99. The method of claim78, wherein said polypeptide is in a pharmaceutically acceptableexcipient in the form of a unit dosage formulation.
 100. The method ofclaim 85, wherein said polypeptide is in a pharmaceutically acceptableexcipient.
 101. The method of claim 85, wherein said polypeptide is in apharmaceutically acceptable excipient suitable for oral administrationto a mammal.
 102. The method of claim 85, wherein said polypeptide is ina pharmaceutically acceptable excipient in the form of a unit dosageformulation.